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Sample records for nicotiana attenuata wrky3

  1. Priming of anti-herbivore defence in Nicotiana attenuata by insect oviposition: herbivore-specific effects.

    PubMed

    Bandoly, Michele; Grichnik, Roland; Hilker, Monika; Steppuhn, Anke

    2016-04-01

    Oviposition by Spodoptera exigua on Nicotiana attenuata primes plant defence against its larvae that consequently suffer reduced performance. To reveal whether this is a general response of tobacco to insect oviposition or species-specific, we investigated whether also Manduca sexta oviposition primes N. attenuata's anti-herbivore defence. The plant response to M. sexta and S. exigua oviposition overlapped in the egg-primed feeding-induced production of the phenylpropanoid caffeoylputrescine. While M. sexta larvae were unaffected in their performance, they showed a novel response to the oviposition-mediated plant changes: a reduced antimicrobial activity in their haemolymph. In a cross-resistance experiment, S. exigua larvae suffered reduced performance on M. sexta-oviposited plants like they did on S. exigua-oviposited plants. The M. sexta oviposition-mediated plant effects on the S. exigua larval performance and on M. sexta larval immunity required expression of the NaMyb8 transcription factor that is governing biosynthesis of phenylpropanoids such as caffeoylputrescine. Thus, NaMyb8-dependent defence traits mediate the effects that oviposition by both lepidopteran species exerts on the plant's anti-herbivore defence. These results suggest that oviposition by lepidopteran species on N. attenuata leaves may generally prime the feeding-induced production of certain plant defence compounds but that different herbivore species show different susceptibility to egg-primed plant effects. PMID:26566692

  2. Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata.

    PubMed

    Kessler, Danny; Bhattacharya, Samik; Diezel, Celia; Rothe, Eva; Gase, Klaus; Schöttner, Matthias; Baldwin, Ian T

    2012-08-01

    Many plants use sophisticated strategies to maximize their reproductive success via outcrossing. Nicotiana attenuata flowers produce nectar with nicotine at concentrations that are repellent to hummingbirds, increasing the number of flowers visited per plant. In choice tests using native hummingbirds, we show that these important pollinators learn to tolerate high-nicotine nectar but prefer low-nicotine nectar, and show no signs of nicotine addiction. Nectar nicotine concentrations, unlike those of other vegetative tissues, are unpredictably variable among flowers, not only among populations, but also within populations, and even among flowers within an inflorescence. To evaluate whether variations in nectar nicotine concentrations increase outcrossing, polymorphic microsatellite markers, optimized to evaluate paternity in native N. attenuata populations, were used to compare outcrossing in plants silenced for expression of a biosynthetic gene for nicotine production (Napmt1/2) and in control empty vector plants, which were antherectomized and transplanted into native populations. When only exposed to hummingbird pollinators, seeds produced by flowers with nicotine in their nectar had a greater number of genetically different sires, compared to seeds from nicotine-free flowers. As the variation in nectar nicotine levels among flowers in an inflorescence decreased in N. attenuata plants silenced in various combinations of three Dicer-like (DCL) proteins, small RNAs are probably involved in the unpredictable variation in nectar nicotine levels within a plant.

  3. Sebacina vermifera promotes the growth and fitness of Nicotiana attenuata by inhibiting ethylene signaling.

    PubMed

    Barazani, Oz; von Dahl, Caroline C; Baldwin, Ian T

    2007-06-01

    Sebacina vermifera, a growth-promoting endophytic fungus, significantly increases Nicotiana attenuata's growth but impairs both its herbivore resistance and its accumulation of the costly, jasmonic acid (JA)-regulated defense protein, trypsin proteinase inhibitor (TPI). To determine if the fungi's growth-promoting effects can be attributed to lower TPI-related defense costs, we inoculated transformed N. attenuata plants silenced in their ability to synthesize JA, JA-isoleucine, and TPI by antisense (lipoxygenase 3 [as-lox3] and Thr deaminase [as-td]) and inverted repeat (ir-tpi) expression, and found that inoculation promoted plant growth as in untransformed wild-type plants. Moreover, herbivore-elicited increases in JA and JA-isoleucine concentrations did not differ between inoculated and uninoculated wild-type plants. However, inoculation significantly reduced the morphological effect of 1-aminocyclopropane-1-carboxylic acid on wild-type seedlings in a triple response assay, suggesting that ethylene signaling was impaired. Furthermore, S. vermifera failed to promote the growth of N. attenuata plants transformed to silence ethylene production (1-aminocyclopropane-1-carboxylic acid oxidase [ir-aco]). Inoculating wild-type plants with S. vermifera decreased the ethylene burst elicited by applying Manduca sexta oral secretions to mechanical wounds. Accordingly, oral secretion-elicited transcript levels of the ethylene synthesis genes NaACS3, NaACO1, and NaACO3 in inoculated plants were significantly lower compared to these levels in uninoculated wild-type plants. Inoculation accelerated germination in wild-type seeds; however, uninoculated wild-type seeds germinated as rapidly as inoculated seeds in the presence of the ethylene scrubber KMnO(4). In contrast, neither inoculation nor KMnO(4) exposure influenced the germination of ir-aco seeds. We conclude that S. vermifera increases plant growth by impairing ethylene production independently of JA signaling and TPI

  4. Unbiased Transcriptional Comparisons of Generalist and Specialist Herbivores Feeding on Progressively Defenseless Nicotiana attenuata Plants

    PubMed Central

    Govind, Geetha; Mittapalli, Omprakash; Griebel, Thasso; Allmann, Silke; Böcker, Sebastian; Baldwin, Ian Thomas

    2010-01-01

    Background Herbivore feeding elicits dramatic increases in defenses, most of which require jasmonate (JA) signaling, and against which specialist herbivores are thought to be better adapted than generalist herbivores. Unbiased transcriptional analyses of how neonate larvae cope with these induced plant defenses are lacking. Methodology/Principal Findings We created cDNA microarrays for Manduca sexta and Heliothis virescens separately, by spotting normalized midgut-specific cDNA libraries created from larvae that fed for 24 hours on MeJA-elicited wild-type (WT) Nicotiana attenuata plants. These microarrays were hybridized with labeled probes from neonates that fed for 24 hours on WT and isogenic plants progressively silenced in JA-mediated defenses (N: nicotine; N/PI: N and trypsin protease inhibitors; JA: all JA-mediated defenses). H. virescens neonates regulated 16 times more genes than did M. sexta neonates when they fed on plants silenced in JA-mediated defenses, and for both species, the greater the number of defenses silenced in the host plant (JA > N/PI > N), the greater were the number of transcripts regulated in the larvae. M. sexta larvae tended to down-regulate while H. virescens larvae up- and down-regulated transcripts from the same functional categories of genes. M. sexta larvae regulated transcripts in a diet-specific manner, while H. virescens larvae regulated a similar suite of transcripts across all diet types. Conclusions/Significance The observations are consistent with the expectation that specialists are better adapted than generalist herbivores to the defense responses elicited in their host plants by their feeding. While M. sexta larvae appear to be better adapted to N. attenuata's defenses, some of the elicited responses remain effective defenses against both herbivore species. The regulated genes provide novel insights into larval adaptations to N. attenuata's induced defenses, and represent potential targets for plant-mediated RNAi to

  5. Shifting Nicotiana attenuata's diurnal rhythm does not alter its resistance to the specialist herbivore Manduca sexta.

    PubMed

    Herden, Jasmin; Meldau, Stefan; Kim, Sang-Gyu; Kunert, Grit; Joo, Youngsung; Baldwin, Ian T; Schuman, Meredith C

    2016-07-01

    Arabidopsis thaliana plants are less resistant to attack by the generalist lepidopteran herbivore Trichoplusia ni when plants and herbivores are entrained to opposite, versus identical diurnal cycles and tested under constant conditions. This effect is associated with circadian fluctuations in levels of jasmonic acid, the transcription factor MYC2, and glucosinolate contents in leaves. We tested whether a similar effect could be observed in a different plant-herbivore system: the wild tobacco Nicotiana attenuata and its co-evolved specialist herbivore, Manduca sexta. We measured larval growth on plants under both constant and diurnal conditions following identical or opposite entrainment, profiled the metabolome of attacked leaf tissue, quantified specific metabolites known to reduce M. sexta growth, and monitored M. sexta feeding activity under all experimental conditions. Entrainment did not consistently affect M. sexta growth or plant defense induction. However, both were reduced under constant dark conditions, as was M. sexta feeding activity. Our data indicate that the response induced by M. sexta in N. attenuata is robust to diurnal cues and independent of plant or herbivore entrainment. We propose that while the patterns of constitutive or general damage-induced defense may undergo circadian fluctuation, the orchestration of specific induced responses is more complex. PMID:26699809

  6. Opportunistic out-crossing in Nicotiana attenuata (Solanaceae), a predominantly self-fertilizing native tobacco

    PubMed Central

    Sime, Karen R; Baldwin, Ian T

    2003-01-01

    Background Although Nicotiana attenuata is entirely self-compatible, chemical and other floral traits suggest selection for the maintenance of advertisement for moth pollinators. Results Experimental exclusions of pollinators from plants with emasculated flowers in natural populations in southern Utah during an outbreak of the hawkmoth Hyles lineata revealed that 24% of the seed set could be attributed to insect pollination, and eliminated wind pollination and apomixis as contributing to seed set. Hence these moths can mediate gene flow when self-pollen is unavailable. To quantify gene flow when self-pollen is available, plants were transformed with two marker genes: hygromycin-B resistance and β-glucuronidase. The utility of these genetic markers to measure gene flow between plants was examined by mixing pollen from plants homozygous for both genes with self-pollen in different ratios and hand-pollinating emasculated flowers of plants growing in a natural population. The proportion of transformed seeds was positively correlated with the amount of transformed pollen applied to stigmas. In glasshouse experiments with the hawkmoth Manduca sexta and experimental arrays of transformed and wild-type plants, pollination mediated by moths accounted for 2.5% of the seed set. Conclusions Even though moth pollination is rare and highly variable for this largely selfing plant, N. attenuata opportunistically employs a mixed-mating system. PMID:12866951

  7. HSPRO controls early Nicotiana attenuata seedling growth during interaction with the fungus Piriformospora indica.

    PubMed

    Schuck, Stefan; Camehl, Iris; Gilardoni, Paola A; Oelmueller, Ralf; Baldwin, Ian T; Bonaventure, Gustavo

    2012-10-01

    In a previous study aimed at identifying regulators of Nicotiana attenuata responses against chewing insects, a 26-nucleotide tag matching the HSPRO (ORTHOLOG OF SUGAR BEET Hs1(pro)(-)(1)) gene was found to be strongly induced after simulated herbivory (Gilardoni et al., 2010). Here we characterized the function of HSPRO during biotic interactions in transgenic N. attenuata plants silenced in its expression (ir-hspro). In wild-type plants, HSPRO expression was not only induced during simulated herbivory but also when leaves were inoculated with Pseudomonas syringae pv tomato DC3000 and roots with the growth-promoting fungus Piriformospora indica. Reduced HSPRO expression did not affect the regulation of direct defenses against Manduca sexta herbivory or P. syringae pv tomato DC3000 infection rates. However, reduced HSPRO expression positively influenced early seedling growth during interaction with P. indica; fungus-colonized ir-hspro seedlings increased their fresh biomass by 30% compared with the wild type. Grafting experiments demonstrated that reduced HSPRO expression in roots was sufficient to induce differential growth promotion in both roots and shoots. This effect was accompanied by changes in the expression of 417 genes in colonized roots, most of which were metabolic genes. The lack of major differences in the metabolic profiles of ir-hspro and wild-type colonized roots (as analyzed by liquid chromatography time-of-flight mass spectrometry) suggested that accelerated metabolic rates were involved. We conclude that HSPRO participates in a whole-plant change in growth physiology when seedlings interact with P. indica. PMID:22892352

  8. Allometric analysis of the induced flavonols on the leaf surface of wild tobacco (Nicotiana attenuata).

    PubMed

    Roda, Amy L; Oldham, Neil J; Svatos, Ales; Baldwin, Ian T

    2003-02-01

    Trichomes excrete secondary metabolites that may alter the chemical composition of the leaf surface, reducing damage caused by herbivores, pathogens and abiotic stresses. We examined the surface exudates produced by Nicotiana attenuata Torr. Ex Wats., a plant known to contain and secrete a number of secondary metabolites that are toxic or a deterrent to herbivorous insects. Extractions specific to the leaf surface, the trichomes, and the laminar components demonstrated the localization of particular compounds. Diterpene glycosides occurred exclusively in leaf mesophyll, whereas nicotine was found in both the trichomes and mesophyll. Neither rutin nor nicotine was found on the leaf surface. Quercetin and 7 methylated derivatives were found in the glandular trichomes and appeared to be excreted onto the leaf surface. We examined the elicitation of these flavonols on the leaf surface with a surface-area allometric analysis, which measures changes in metabolites independent of the effects of leaf expansion. The flavonols responded differently to wounding, methyl jasmonate (MeJA), herbivore attack and UV-C radiation, and the response patterns corresponded to their compound-specific allometries. Finding greater amounts of quercetin on younger leaves and reduced amounts after herbivore feeding and MeJA treatment, we hypothesized that quercetin may function as an attractant, helping the insects locate a preferred feeding site. Consistent with this hypothesis, mirids (Tupiocoris notatus) were found more often on mature leaves sprayed with quercetin at a concentration typical of young leaves than on unsupplemented mature leaves. The composition of metabolites on the leaf surface of N. attenuata changes throughout leaf development and in response to herbivore attack or environmental stress, and these changes are mediated in part by responses of the glandular trichomes.

  9. Phenotypic, genetic and genomic consequences of natural and synthetic polyploidization of Nicotiana attenuata and Nicotiana obtusifolia

    PubMed Central

    Anssour, S.; Krügel, T.; Sharbel, T. F.; Saluz, H. P.; Bonaventure, G.; Baldwin, I. T.

    2009-01-01

    Background and Methods Polyploidy results in genetic turmoil, much of which is associated with new phenotypes that result in speciation. Five independent lines of synthetic allotetraploid N. × obtusiata (N × o) were created from crosses between the diploid N. attenuata (Na) (♂) and N. obtusifolia (No) (♀) and the autotetraploids of Na (NaT) and No (NoT) were synthesized. Their genetic, genomic and phenotypic changes were then compared with those of the parental diploid species (Na and No) as well as to the natural allotetraploids, N. quadrivalvis (Nq) and N. clevelandii (Nc), which formed 1 million years ago from crosses between ancient Na and No. Key Results DNA fingerprinting profiles (by UP-PCR) revealed that the five N × o lines shared similar but not identical profiles. Both synthetic and natural polyploidy showed a dosage effect on genome size (as measured in seeds); however, only Nq was associated with a genome upsizing. Phenotypic analysis revealed that at the cellular level, N × o lines had phenotypes intermediate of the parental phenotypes. Both allo- and autotetraploidization had a dosage effect on seed and dry biomass (except for NaT), but not on stalk height at first flower. Nc showed paternal (Na) cellular phenotypes but inherited maternal (No) biomass and seed mass, whereas Nq showed maternal (No) cellular phenotypes but inherited paternal (Na) biomass and seed mass patterns. Principal component analysis grouped Nq with N × o lines, due to similar seed mass, stalk height and genome size. These traits separated Nc, No and Na from Nq and N × o lines, whereas biomass distinguished Na from N × o and Nq lines, and NaT clustered closer to Nq and N × o lines than to Na. Conclusions Both allo- and autotetraploidy induce considerable morphological, genetic and genomic changes, many of which are retained by at least one of the natural polyploids. It is proposed that both natural and synthetic polyploids are well suited for studying the evolution of

  10. Insect herbivory elicits genome-wide alternative splicing responses in Nicotiana attenuata.

    PubMed

    Ling, Zhihao; Zhou, Wenwu; Baldwin, Ian T; Xu, Shuqing

    2015-10-01

    Changes in gene expression and alternative splicing (AS) are involved in many responses to abiotic and biotic stresses in eukaryotic organisms. In response to attack and oviposition by insect herbivores, plants elicit rapid changes in gene expression which are essential for the activation of plant defenses; however, the herbivory-induced changes in AS remain unstudied. Using mRNA sequencing, we performed a genome-wide analysis on tobacco hornworm (Manduca sexta) feeding-induced AS in both leaves and roots of Nicotiana attenuata. Feeding by M. sexta for 5 h reduced total AS events by 7.3% in leaves but increased them in roots by 8.0% and significantly changed AS patterns in leaves and roots of existing AS genes. Feeding by M. sexta also resulted in increased (in roots) and decreased (in leaves) transcript levels of the serine/arginine-rich (SR) proteins that are involved in the AS machinery of plants and induced changes in SR gene expression that were jasmonic acid (JA)-independent in leaves but JA-dependent in roots. Changes in AS and gene expression elicited by M. sexta feeding were regulated independently in both tissues. This study provides genome-wide evidence that insect herbivory induces changes not only in the levels of gene expression but also in their splicing, which might contribute to defense against and/or tolerance of herbivory. PMID:26306554

  11. Insect herbivory elicits genome-wide alternative splicing responses in Nicotiana attenuata.

    PubMed

    Ling, Zhihao; Zhou, Wenwu; Baldwin, Ian T; Xu, Shuqing

    2015-10-01

    Changes in gene expression and alternative splicing (AS) are involved in many responses to abiotic and biotic stresses in eukaryotic organisms. In response to attack and oviposition by insect herbivores, plants elicit rapid changes in gene expression which are essential for the activation of plant defenses; however, the herbivory-induced changes in AS remain unstudied. Using mRNA sequencing, we performed a genome-wide analysis on tobacco hornworm (Manduca sexta) feeding-induced AS in both leaves and roots of Nicotiana attenuata. Feeding by M. sexta for 5 h reduced total AS events by 7.3% in leaves but increased them in roots by 8.0% and significantly changed AS patterns in leaves and roots of existing AS genes. Feeding by M. sexta also resulted in increased (in roots) and decreased (in leaves) transcript levels of the serine/arginine-rich (SR) proteins that are involved in the AS machinery of plants and induced changes in SR gene expression that were jasmonic acid (JA)-independent in leaves but JA-dependent in roots. Changes in AS and gene expression elicited by M. sexta feeding were regulated independently in both tissues. This study provides genome-wide evidence that insect herbivory induces changes not only in the levels of gene expression but also in their splicing, which might contribute to defense against and/or tolerance of herbivory.

  12. Herbivore induction of jasmonic acid and chemical defences reduce photosynthesis in Nicotiana attenuata.

    PubMed

    Nabity, Paul D; Zavala, Jorge A; DeLucia, Evan H

    2013-01-01

    Herbivory initiates a shift in plant metabolism from growth to defence that may reduce fitness in the absence of further herbivory. However, the defence-induced changes in carbon assimilation that precede this reallocation in resources remain largely undetermined. This study characterized the response of photosynthesis to herbivore induction of jasmonic acid (JA)-related defences in Nicotiana attenuata to increase understanding of these mechanisms. It was hypothesized that JA-induced defences would immediately reduce the component processes of photosynthesis upon attack and was predicted that wild-type plants would suffer greater reductions in photosynthesis than plants lacking JA-induced defences. Gas exchange, chlorophyll fluorescence, and thermal spatial patterns were measured together with the production of defence-related metabolites after attack and through recovery. Herbivore damage immediately reduced electron transport and gas exchange in wild-type plants, and gas exchange remained suppressed for several days after attack. The sustained reductions in gas exchange occurred concurrently with increased defence metabolites in wild-type plants, whereas plants lacking JA-induced defences suffered minimal suppression in photosynthesis and no increase in defence metabolite production. This suppression in photosynthesis occurred only after sustained defence signalling and defence chemical mobilization, whereas a short bout of feeding damage only transiently altered components of photosynthesis. It was identified that lipoxygenase signalling interacted with photosynthetic electron transport and that the resulting JA-related metabolites reduced photosynthesis. These data represent a metabolic cost to mounting a chemical defence against herbivory and link defence-signalling networks to the differential effects of herbivory on photosynthesis in remaining leaf tissues in a time-dependent manner.

  13. Jasmonic acid signalling mediates resistance of the wild tobacco Nicotiana attenuata to its native Fusarium, but not Alternaria, fungal pathogens.

    PubMed

    Luu, Van Thi; Schuck, Stefan; Kim, Sang-Gyu; Weinhold, Arne; Baldwin, Ian T

    2015-03-01

    We recently characterized a highly dynamic fungal disease outbreak in native populations of Nicotiana attenuata in the southwestern United States. Here, we explore how phytohormone signalling contributes to the observed disease dynamics. Single inoculation with three native Fusarium and Alternaria fungal pathogens, isolated from diseased plants growing in native populations, resulted in disease symptoms characteristic for each pathogen species. While Alternaria sp.-infected plants displayed fewer symptoms and recovered, Fusarium spp.-infected plants became chlorotic and frequently spontaneously wilted. Jasmonic acid (JA) and salicylic acid (SA) levels were differentially induced after Fusarium or Alternaria infection. Transgenic N. attenuata lines silenced in JA production or JA conjugation to isoleucine (JA-Ile), but not in JA perception, were highly susceptible to infection by F. brachygibbosum Utah 4, indicating that products derived from the JA-Ile biosynthetic pathway, but not their perception, is associated with increased Fusarium resistance. Infection assays using ov-nahG plants which were silenced in pathogen-induced SA accumulations revealed that SA may increase N. attenuata's resistance to Fusarium infection but not to Alternaria. Taken together, we propose that the dynamics of fungal disease symptoms among plants in native populations may be explained by a complex interplay of phytohormone responses to attack by multiple pathogens.

  14. Jasmonoyl-L-isoleucine coordinates metabolic networks required for anthesis and floral attractant emission in wild tobacco (Nicotiana attenuata).

    PubMed

    Stitz, Michael; Hartl, Markus; Baldwin, Ian T; Gaquerel, Emmanuel

    2014-10-01

    Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-L-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study.

  15. Differences in Nicotine Metabolism of Two Nicotiana attenuata Herbivores Render Them Differentially Susceptible to a Common Native Predator

    PubMed Central

    Kumar, Pavan; Rathi, Preeti; Schöttner, Matthias; Baldwin, Ian T.; Pandit, Sagar

    2014-01-01

    Background Nicotiana attenuata is attacked by larvae of both specialist (Manduca sexta) and generalist (Spodoptera exigua) lepidopteran herbivores in its native habitat. Nicotine is one of N. attenuata's important defenses. M. sexta is highly nicotine tolerant; whether cytochrome P450 (CYP)-mediated oxidative detoxification and/or rapid excretion is responsible for its exceptional tolerance remains unknown despite five decades of study. Recently, we demonstrated that M. sexta uses its nicotine-induced CYP6B46 to efflux midgut-nicotine into the hemolymph, facilitating nicotine exhalation that deters predatory wolf spiders (Camptocosa parallela). S. exigua's nicotine metabolism is uninvestigated. Methodology/Principal Findings We compared the ability of these two herbivores to metabolize, tolerate and co-opt ingested nicotine for defense against the wolf spider. In addition, we analyzed the spider's excretion to gain insights into its nicotine metabolism. Contrary to previous reports, we found that M. sexta larvae neither accumulate the common nicotine oxides (cotinine, cotinine N-oxide and nicotine N-oxide) nor excrete them faster than nicotine. In M. sexta larvae, ingestion of nicotine as well as its oxides increases the accumulation of CYP6B46 transcripts. In contrast, S. exigua accumulates nicotine oxides and exhales less (66%) nicotine than does M. sexta. Spiders prefer nicotine-fed S. exigua over M. sexta, a preference reversed by topical or headspace nicotine supplementation, but not ingested or topically-coated nicotine oxides, suggesting that externalized nicotine but not the nicotine detoxification products deter spider predation. The spiders also do not accumulate nicotine oxides. Conclusions Nicotine oxidation reduces S. exigua's headspace-nicotine and renders it more susceptible to predation by spiders than M. sexta, which exhales unmetabolized nicotine. These results are consistent with the hypothesis that generalist herbivores incur costs of

  16. Isolating Fungal Pathogens from a Dynamic Disease Outbreak in a Native Plant Population to Establish Plant-Pathogen Bioassays for the Ecological Model Plant Nicotiana attenuata

    PubMed Central

    Schuck, Stefan; Baldwin, Ian T.

    2014-01-01

    The wild tobacco species Nicotiana attenuata has been intensively used as a model plant to study its interaction with insect herbivores and pollinators in nature, however very little is known about its native pathogen community. We describe a fungal disease outbreak in a native N. attenuata population comprising 873 plants growing in an area of about 1500 m2. The population was divided into 14 subpopulations and disease symptom development in the subpopulations was monitored for 16 days, revealing a waxing and waning of visible disease symptoms with some diseased plants recovering fully. Native fungal N. attenuata pathogens were isolated from diseased plants, characterized genetically, chemotaxonomically and morphologically, revealing several isolates of the ascomycete genera Fusarium and Alternaria, that differed in the type and strength of the disease symptoms they caused in bioassays on either detached leaves or intact soil-grown plants. These isolates and the bioassays will empower the study of N. attenuata-pathogen interactions in a realistic ecological context. PMID:25036191

  17. Silencing of hydroperoxide lyase and allene oxide synthase reveals substrate and defense signaling crosstalk in Nicotiana attenuata.

    PubMed

    Halitschke, Rayko; Ziegler, Jörg; Keinänen, Markku; Baldwin, Ian T

    2004-10-01

    The fatty acid hydroperoxide (HP) substrates required for the biosynthesis of jasmonic acid (JA) and green leaf volatiles (GLVs) are supplied by separate lipoxygenases (LOX). We silenced the expression of two genes downstream of the LOX: allene oxide synthase (AOS) and HP lyase (HPL) by antisense expression of endogenous genes (NaAOS, NaHPL) in Nicotiana attenuata, in which the biosynthesis of JA is amplified by herbivore-specific elicitors. We report that these elicitors also amplify wound-induced GLV releases, but suppress the wound-induced increase of NaHPL transcripts, suggesting that substrate flux controls GLV biosynthesis. As expected, silencing of NaHPL and NaAOS reduced GLV release and JA accumulation, respectively. Surprisingly, HPL- and AOS-silenced plants had enhanced JA and GLV responses, suggesting substrate 'crosstalk' between these two oxylipin cascades. Plants with depleted GLVs (as-hpl) were less attractive than wild type (WT) or empty vector control plants in choice-tests with native lepidopteran herbivores. In feeding trials, Manduca sexta larvae developed slower on as-hpl plants. The reduced larval consumption and performance, which was not caused by increases in defense responses in as-hpl plants, could be restored to WT levels by the addition of synthetic GLVs, demonstrating that GLVs function as feeding stimulants. Gene expression profiling by cDNA microarray analysis and characterization of several induced defenses in herbivore-elicited as-hpl and as-aos plants revealed differential involvement of JA and GLVs in defense signaling. Elicitation of volatile terpenoids (an indirect defense) requires JA signaling, where as trypsin protease inhibitor elicitation (a direct defense) requires both functional JA and GLV cascades.

  18. MYB8 Controls Inducible Phenolamide Levels by Activating Three Novel Hydroxycinnamoyl-Coenzyme A:Polyamine Transferases in Nicotiana attenuata[W][OA

    PubMed Central

    Onkokesung, Nawaporn; Gaquerel, Emmanuel; Kotkar, Hemlata; Kaur, Harleen; Baldwin, Ian T.; Galis, Ivan

    2012-01-01

    A large number of plants accumulate N-acylated polyamines (phenolamides [PAs]) in response to biotic and/or abiotic stress conditions. In the native tobacco (Nicotiana attenuata), the accumulation of two major PAs, caffeoylputrescine and dicaffeoylspermidine (DCS), after herbivore attack is known to be controlled by a key transcription factor, MYB8. Using a broadly targeted metabolomics approach, we show that a much larger spectrum of PAs composed of hydroxycinnamic acids and two polyamines, putrescine and spermidine, is regulated by this transcription factor. We cloned several novel MYB8-regulated genes, annotated as putative acyltransferases, and analyzed their function. One of the novel acyltransferases (AT1) is shown to encode a hydroxycinnamoyl-coenzyme A:putrescine acyltransferase responsible for caffeoylputrescine biosynthesis in tobacco. Another gene (acyltransferase DH29), specific for spermidine conjugation, mediates the initial acylation step in DCS formation. Although this enzyme was not able to perform the second acylation toward DCS biosynthesis, another acyltransferase gene, CV86, proposed to act on monoacylated spermidines, was isolated and partially characterized. The activation of MYB8 in response to herbivore attack and associated signals required the activity of LIPOXYGENASE3, a gene involved in jasmonic acid (JA) biosynthesis in N. attenuata. These new results allow us to reconstruct a complete branch in JA signaling that defends N. attenuata plants against herbivores: JA via MYB8’s transcriptional control of AT1 and DH29 genes controls the entire branch of PA biosynthesis, which allows N. attenuata to mount a chemically diverse (and likely efficient) defense shield against herbivores. PMID:22082505

  19. Ectopic Terpene Synthase Expression Enhances Sesquiterpene Emission in Nicotiana attenuata without Altering Defense or Development of Transgenic Plants or Neighbors1[W

    PubMed Central

    Schuman, Meredith C.; Palmer-Young, Evan C.; Schmidt, Axel; Gershenzon, Jonathan; Baldwin, Ian T.

    2014-01-01

    Sesquiterpenoids, with approximately 5,000 structures, are the most diverse class of plant volatiles with manifold hypothesized functions in defense, stress tolerance, and signaling between and within plants. These hypotheses have often been tested by transforming plants with sesquiterpene synthases expressed behind the constitutively active 35S promoter, which may have physiological costs measured as inhibited growth and reduced reproduction or may require augmentation of substrate pools to achieve enhanced emission, complicating the interpretation of data from affected transgenic lines. Here, we expressed maize (Zea mays) terpene synthase10 (ZmTPS10), which produces (E)-α-bergamotene and (E)-β-farnesene, or a point mutant ZmTPS10M, which produces primarily (E)-β-farnesene, under control of the 35S promoter in the ecological model plant Nicotiana attenuata. Transgenic N. attenuata plants had specifically enhanced emission of target sesquiterpene(s) with no changes detected in their emission of any other volatiles. Treatment with herbivore or jasmonate elicitors induces emission of (E)-α-bergamotene in wild-type plants and also tended to increase emission of (E)-α-bergamotene and (E)-β-farnesene in transgenics. However, transgenics did not differ from the wild type in defense signaling or chemistry and did not alter defense chemistry in neighboring wild-type plants. These data are inconsistent with within-plant and between-plant signaling functions of (E)-β-farnesene and (E)-α-bergamotene in N. attenuata. Ectopic sesquiterpene emission was apparently not costly for transgenics, which were similar to wild-type plants in their growth and reproduction, even when forced to compete for common resources. These transgenics would be well suited for field experiments to investigate indirect ecological effects of sesquiterpenes for a wild plant in its native habitat. PMID:25187528

  20. The HERBIVORE ELICITOR-REGULATED1 Gene Enhances Abscisic Acid Levels and Defenses against Herbivores in Nicotiana attenuata Plants1[C][W][OPEN

    PubMed Central

    Dinh, Son Truong; Baldwin, Ian T.; Galis, Ivan

    2013-01-01

    Nicotiana attenuata plants can distinguish the damage caused by herbivore feeding from other types of damage by perceiving herbivore-associated elicitors, such as the fatty acid-amino acid conjugates (FACs) in oral secretions (OS) of Manduca sexta larvae, which are introduced into wounds during feeding. However, the transduction of FAC signals into downstream plant defense responses is still not well established. We identified a novel FAC-regulated protein in N. attenuata (NaHER1; for herbivore elicitor regulated) and show that it is an indispensable part of the OS signal transduction pathway. N. attenuata plants silenced in the expression of NaHER1 by RNA interference (irHER1) were unable to amplify their defenses beyond basal, wound-induced levels in response to OS elicitation. M. sexta larvae performed 2-fold better when reared on irHER1 plants, which released less volatile organic compounds (indirect defense) and had strongly reduced levels of several direct defense metabolites, including trypsin proteinase inhibitors, 17-hydroxygeranyllinallool diterpene glycosides, and caffeoylputrescine, after real and/or simulated herbivore attack. In parallel to impaired jasmonate signaling and metabolism, irHER1 plants were more drought sensitive and showed reduced levels of abscisic acid (ABA) in the leaves, suggesting that silencing of NaHER1 interfered with ABA metabolism. Because treatment of irHER1 plants with ABA results in both the accumulation of significantly more ABA catabolites and the complete restoration of normal wild-type levels of OS-induced defense metabolites, we conclude that NaHER1 acts as a natural suppressor of ABA catabolism after herbivore attack, which, in turn, activates the full defense profile and resistance against herbivores. PMID:23784463

  1. Jasmonoyl-l-Isoleucine Coordinates Metabolic Networks Required for Anthesis and Floral Attractant Emission in Wild Tobacco (Nicotiana attenuata)[C][W][OPEN

    PubMed Central

    Stitz, Michael; Hartl, Markus; Baldwin, Ian T.; Gaquerel, Emmanuel

    2014-01-01

    Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-l-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study. PMID:25326292

  2. Up in smoke: I. Smoke-derived germination cues for postfire annual,Nicotiana attenuata torr. Ex. Watson.

    PubMed

    Baldwin, I T; Staszak-Kozinski, L; Davidson, R

    1994-09-01

    Some postfire annuals with dormant seeds use heat or chemical cues from charred wood to synchronize their germination with the postfire environment. We report that wood smoke and polar extracts of wood smoke, but not the ash of burned wood, contain potent cue(s) that stimulate germination in the postfire annual plant,Nicotiana attenuata. We examined the responses of seeds from six populations of plants from southwest Utah to extracts of smoke and found the proportion of viable seeds that germinated in the presence of smoke cues to vary between populations but to be consistent between generations. With the most dormant genotypes, we examine three mechanisms by which smoke-derived chemical cues may stimulate germination (chemical scarification of the seed coat and nutritive- and signal-mediated stimulation of germination) and report that the response is consistent with the signal-mediated mechanism. The germination cue(s) found in smoke are produced by the burning of hay, hardwood branches, leaves, and, to a lesser degree, cellulose. Moreover, the cues are found in the common food condiment, "liquid smoke," and we find no significant differences between brands. With a bioassay-driven fractionation of liquid smoke, we identified 71 compounds in active fractions by GC-MS and AA spectrometry. However, when these compounds were tested in pure form or in combinations that mimicked the composition of active fractions over a range of concentrations, they failed to stimulate germination to the same degree that smoke fractions did. Moreover, enzymatic oxidation of some of these compounds also failed to stimulate germination. In addition, we tested 43 additional compounds also reported from smoke, 85 compounds that were structurally similar to those reported from smoke and 34 compounds reported to influence germination in other species. Of the 233 compounds tested, 16 proved to inhibit germination at the concentrations tested, and none reproduced the activity of wood smoke. By

  3. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis

    PubMed Central

    Groten, Karin; Pahari, Nabin T.; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T.

    2015-01-01

    Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large

  4. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis.

    PubMed

    Groten, Karin; Pahari, Nabin T; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T

    2015-01-01

    Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large

  5. Up in smoke: II. Germination ofNicotiana attenuata in response to smoke-derived cues and nutrients in burned and unburned soils.

    PubMed

    Baldwin, I T; Morse, L

    1994-09-01

    Nicotiana attenuata is a native tobacco that is commonly found usually one growing season after fires in the blackbrush, sagebrush and pinyon-juniper forests of the Great Basin desert of North America. This plant also occurs in isolated dry washes and roadsides for many consecutive seasons. Postfire annuals are thought to synchronize their germination from the seed bank with the postfire environment in response to increases in (1) fire-related cues or (2) nutrient supply rates resulting from the mineralization of nutrients by fire, or (3) the removal of allelochemicals produced by the dominant vegetation occupying the sites before the burn or the microbial community associated with the dominant vegetation. We examine the effect of these three changes on the germination ofN. attenuata seed from artificial seed banks made with burned and unburned soil taken in 1993 from under four dominant shrub species (Coleogyne ramosissima, Yucca baccata, Lycium andersonnii, Purshia tridentata) of an area that burned in 1992 and from two dry washes in whichN. attenuata populations have persisted since at least 1988. We utilize our recent discovery that aqueous extracts of wood smoke contain potent germination cue(s) for this species and the established observation that nitrate stimulates germination in manyNicotiana species. In two experiments, we added smoke-derived germination cues and nutrients separately and in combinations to the artificial seed banks, measured germination rates, and inferred the effect of burning by the response of the seed banks to these additions. Germination rates of seed in burned soil were consistently higher than those in unburned soil collected from under all species tested; concentrations of nitrate, P, Mn, and Ca were also higher in burned than unburned soils. Because the addition of more cue and nitrate to burned soil increased germination rates, these soil components may not be at concentrations sufficient to saturate the germination response one

  6. Up in smoke: II. Germination ofNicotiana attenuata in response to smoke-derived cues and nutrients in burned and unburned soils.

    PubMed

    Baldwin, I T; Morse, L

    1994-09-01

    Nicotiana attenuata is a native tobacco that is commonly found usually one growing season after fires in the blackbrush, sagebrush and pinyon-juniper forests of the Great Basin desert of North America. This plant also occurs in isolated dry washes and roadsides for many consecutive seasons. Postfire annuals are thought to synchronize their germination from the seed bank with the postfire environment in response to increases in (1) fire-related cues or (2) nutrient supply rates resulting from the mineralization of nutrients by fire, or (3) the removal of allelochemicals produced by the dominant vegetation occupying the sites before the burn or the microbial community associated with the dominant vegetation. We examine the effect of these three changes on the germination ofN. attenuata seed from artificial seed banks made with burned and unburned soil taken in 1993 from under four dominant shrub species (Coleogyne ramosissima, Yucca baccata, Lycium andersonnii, Purshia tridentata) of an area that burned in 1992 and from two dry washes in whichN. attenuata populations have persisted since at least 1988. We utilize our recent discovery that aqueous extracts of wood smoke contain potent germination cue(s) for this species and the established observation that nitrate stimulates germination in manyNicotiana species. In two experiments, we added smoke-derived germination cues and nutrients separately and in combinations to the artificial seed banks, measured germination rates, and inferred the effect of burning by the response of the seed banks to these additions. Germination rates of seed in burned soil were consistently higher than those in unburned soil collected from under all species tested; concentrations of nitrate, P, Mn, and Ca were also higher in burned than unburned soils. Because the addition of more cue and nitrate to burned soil increased germination rates, these soil components may not be at concentrations sufficient to saturate the germination response one

  7. Jasmonate and ppHsystemin Regulate Key Malonylation Steps in the Biosynthesis of 17-Hydroxygeranyllinalool Diterpene Glycosides, an Abundant and Effective Direct Defense against Herbivores in Nicotiana attenuata[W

    PubMed Central

    Heiling, Sven; Schuman, Meredith C.; Schoettner, Matthias; Mukerjee, Purba; Berger, Beatrice; Schneider, Bernd; Jassbi, Amir R.; Baldwin, Ian T.

    2010-01-01

    We identified 11 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) that occur in concentrations equivalent to starch (mg/g fresh mass) in aboveground tissues of coyote tobacco (Nicotiana attenuata) and differ in their sugar moieties and malonyl sugar esters (0-2). Concentrations of HGL-DTGs, particularly malonylated compounds, are highest in young and reproductive tissues. Within a tissue, herbivore elicitation changes concentrations and biosynthetic kinetics of individual compounds. Using stably transformed N. attenuata plants silenced in jasmonate production and perception, or production of N. attenuata Hyp-rich glycopeptide systemin precursor by RNA interference, we identified malonylation as the key biosynthetic step regulated by herbivory and jasmonate signaling. We stably silenced N. attenuata geranylgeranyl diphosphate synthase (ggpps) to reduce precursors for the HGL-DTG skeleton, resulting in reduced total HGL-DTGs and greater vulnerability to native herbivores in the field. Larvae of the specialist tobacco hornworm (Manduca sexta) grew up to 10 times as large on ggpps silenced plants, and silenced plants suffered significantly more damage from herbivores in N. attenuata's native habitat than did wild-type plants. We propose that high concentrations of HGL-DTGs effectively defend valuable tissues against herbivores and that malonylation may play an important role in regulating the distribution and storage of HGL-DTGs in plants. PMID:20081114

  8. Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions

    PubMed Central

    Ré, Delfina A.; Dezar, Carlos A.; Chan, Raquel L.; Baldwin, Ian T.; Bonaventure, Gustavo

    2011-01-01

    Homeodomain-leucine zipper type I (HD-Zip I) proteins are plant-specific transcription factors associated with the regulation of growth and development in response to changes in the environment. Nicotiana attenuata NaHD20 was identified as an HD-Zip I-coding gene whose expression was induced by multiple stress-associated stimuli including drought and wounding. To study the role of NaHD20 in the integration of stress responses with changes in growth and development, its expression was silenced by virus-induced gene silencing (VIGS), and control and silenced plants were metabolically and developmentally characterized. Phytohormone profiling showed that NaHD20 plays a positive role in abscisic acid (ABA) accumulation in leaves during water stress and in the expression of some dehydration-responsive genes including ABA biosynthetic genes. Moreover, consistent with the high levels of NaHD20 expression in corollas, the emission of benzylacetone from flowers was reduced in NaHD20-silenced plants. Additionally, bolting time and the opening of the inflorescence buds was decelerated in these plants in a specific developmental stage without affecting the total number of flowers produced. Water stress potentiated these effects; however, after plants recovered from this condition, the opening of the inflorescence buds was accelerated in NaHD20-silenced plants. In summary, NaHD20 plays multiple roles in N. attenuata and among these are the coordination of responses to dehydration and its integration with changes in flower transitions. PMID:20713465

  9. The Sesquiterpenes(E)-ß-Farnesene and (E)-α-Bergamotene Quench Ozone but Fail to Protect the Wild Tobacco Nicotiana attenuata from Ozone, UVB, and Drought Stresses.

    PubMed

    Palmer-Young, Evan C; Veit, Daniel; Gershenzon, Jonathan; Schuman, Meredith C

    2015-01-01

    Among the terpenes, isoprene (C5) and monoterpene hydrocarbons (C10) have been shown to ameliorate abiotic stress in a number of plant species via two proposed mechanisms: membrane stabilization and direct antioxidant effects. Sesquiterpene hydrocarbons (C15) not only share the structural properties thought to lend protective qualities to isoprene and monoterpene hydrocarbons, but also react rapidly with ozone, suggesting that sesquiterpenes may similarly enhance tolerance of abiotic stresses. To test whether sesquiterpenes protect plants against ozone, UVB light, or drought, we used transgenic lines of the wild tobacco Nicotiana attenuata. The transgenic plants expressed a maize terpene synthase gene (ZmTPS10) which produced a blend of (E)-ß-farnesene and (E)-α-bergamotene, or a point mutant of the same gene (ZmTPS10M) which produced (E)-ß-farnesene alone,. (E)-ß-farnesene exerted a local, external, and transient ozone-quenching effect in ozone-fumigated chambers, but we found no evidence that enhanced sesquiterpene production by the plant inhibited oxidative damage, or maintained photosynthetic function or plant fitness under acute or chronic stress. Although the sesquiterpenes (E)-ß-farnesene and (E)-α-bergamotene might confer benefits under intermittent heat stress, which was not tested, any roles in relieving abiotic stress may be secondary to their previously demonstrated functions in biotic interactions. PMID:26030663

  10. Herbivore-induced plant vaccination. Part I. The orchestration of plant defenses in nature and their fitness consequences in the wild tobacco Nicotiana attenuata.

    PubMed

    Kessler, André; Baldwin, Ian T

    2004-05-01

    A plant's responses to attack from particular pathogens and herbivores may result in resistance to subsequent attack from the same species, but may also affect different species. Such a cross-resistance, called immunization or vaccination, can benefit the plant, if the fitness consequences of attack from the initial attacker are less than those from subsequent attackers. Here, we report an example of naturally occurring vaccination of the native tobacco plant, Nicotiana attenuata, against Manduca hornworms by prior attack from the mirid bug, Tupiocoris notatus (Dicyphus minimus), which results from the elicitation of two categories of induced plant responses. First, attack from both herbivore species causes the plants in nature to release predator-attracting volatile organic compounds (VOCs), and the attracted generalist predator, Geocoris pallens, preferentially attacks the less mobile hornworm larvae. Second, attack from both mirids and hornworms increases the accumulation of secondary metabolites and proteinase inhibitors (PIs) in the leaf tissue, which is correlated with the slow growth of Manduca larvae. Mirid damage does not significantly reduce the fitness of the plant in nature, whereas attack from the hornworm reduces lifetime seed production. Consequently, plants that are attacked by mirids realize a significant fitness advantage in environments with both herbivores. The combination of growth-slowing direct defenses and predator-attracting indirect defenses results in greater hornworm mortality on mirid-attacked plants and provides the mechanism of the vaccination phenomenon.

  11. A Nicotiana attenuata cell wall invertase inhibitor (NaCWII) reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants.

    PubMed

    Ferrieri, Abigail P; Arce, Carla C M; Machado, Ricardo A R; Meza-Canales, Ivan D; Lima, Eraldo; Baldwin, Ian T; Erb, Matthias

    2015-10-01

    Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source-sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory.

  12. The Sesquiterpenes(E)-ß-Farnesene and (E)-α-Bergamotene Quench Ozone but Fail to Protect the Wild Tobacco Nicotiana attenuata from Ozone, UVB, and Drought Stresses

    PubMed Central

    Palmer-Young, Evan C.; Veit, Daniel; Gershenzon, Jonathan; Schuman, Meredith C.

    2015-01-01

    Among the terpenes, isoprene (C5) and monoterpene hydrocarbons (C10) have been shown to ameliorate abiotic stress in a number of plant species via two proposed mechanisms: membrane stabilization and direct antioxidant effects. Sesquiterpene hydrocarbons (C15) not only share the structural properties thought to lend protective qualities to isoprene and monoterpene hydrocarbons, but also react rapidly with ozone, suggesting that sesquiterpenes may similarly enhance tolerance of abiotic stresses. To test whether sesquiterpenes protect plants against ozone, UVB light, or drought, we used transgenic lines of the wild tobacco Nicotiana attenuata. The transgenic plants expressed a maize terpene synthase gene (ZmTPS10) which produced a blend of (E)-ß-farnesene and (E)-α-bergamotene, or a point mutant of the same gene (ZmTPS10M) which produced (E)-ß-farnesene alone,. (E)-ß-farnesene exerted a local, external, and transient ozone-quenching effect in ozone-fumigated chambers, but we found no evidence that enhanced sesquiterpene production by the plant inhibited oxidative damage, or maintained photosynthetic function or plant fitness under acute or chronic stress. Although the sesquiterpenes (E)-ß-farnesene and (E)-α-bergamotene might confer benefits under intermittent heat stress, which was not tested, any roles in relieving abiotic stress may be secondary to their previously demonstrated functions in biotic interactions. PMID:26030663

  13. A Nicotiana attenuata cell wall invertase inhibitor (NaCWII) reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants.

    PubMed

    Ferrieri, Abigail P; Arce, Carla C M; Machado, Ricardo A R; Meza-Canales, Ivan D; Lima, Eraldo; Baldwin, Ian T; Erb, Matthias

    2015-10-01

    Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source-sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory. PMID:26017581

  14. Tissue Specific Diurnal Rhythms of Metabolites and Their Regulation during Herbivore Attack in a Native Tobacco, Nicotiana attenuata

    PubMed Central

    Kim, Sang-Gyu; Gulati, Jyotasana; Baldwin, Ian T.

    2011-01-01

    Ecological performance is all about timing and the endogenous clock that allows the entrainment of rhythms and anticipation of fitness-determining events is being rapidly characterized. How plants anticipate daily abiotic stresses, such as cold in early mornings and drought at noon, as well as biotic stresses, such as the timing of pathogen infections, is being explored, but little is known about the clock's role in regulating responses to insect herbivores and mutualists, whose behaviors are known to be strongly diurnally regulated and whose attack is known to reconfigure plant metabolomes. We developed a liquid chromatography-mass spectrometry procedure and analyzed its output with model-based peak picking algorithms to identify metabolites with diurnal accumulation patterns in sink/source leaves and roots in an unbiased manner. The response of metabolites with strong diurnal patterns to simulated attack from the specialist herbivore, Manduca sexta larvae was analyzed and annotated with in-house and public databases. Roots and leaves had largely different rhythms and only 10 ions of 182 oscillating ions in leaves and 179 oscillating ions in roots were rhythmic in both tissues: root metabolites mainly peaked at dusk or night, while leaf metabolites peaked during the day. Many oscillating metabolites showed tissue-specific regulation by simulated herbivory of which systemic responses in unattacked tissues were particularly pronounced. Diurnal and herbivory-elicited accumulation patterns of disaccharide, phenylalanine, tyrosine, lyciumoside I, coumaroyl tyramine, 12-oxophytodienoic acid and jasmonic acid and those of their related biosynthetic transcripts were examined in detail. We conclude that oscillating metabolites of N. attenuata accumulate in a highly tissue-specific manner and the patterns reveal pronounced diurnal rhythms in the generalized and specialized metabolism that mediates the plant's responses to herbivores and mutualists. We propose that diurnal

  15. Environmental Stresses of Field Growth Allow Cinnamyl Alcohol Dehydrogenase-Deficient Nicotiana attenuata Plants to Compensate for their Structural Deficiencies1[C][W][OA

    PubMed Central

    Kaur, Harleen; Shaker, Kamel; Heinzel, Nicolas; Ralph, John; Gális, Ivan; Baldwin, Ian T.

    2012-01-01

    The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants. PMID:22645069

  16. NaRALF, a peptide signal essential for the regulation of root hair tip apoplastic pH in Nicotiana attenuata, is required for root hair development and plant growth in native soils.

    PubMed

    Wu, Jinsong; Kurten, Erin L; Monshausen, Gabriele; Hummel, Grégoire M; Gilroy, Simon; Baldwin, Ian T

    2007-12-01

    Rapid alkalinization factor (RALF) is a 49-amino-acid peptide that rapidly alkalinizes cultivated tobacco cell cultures. In the native tobacco Nicotiana attenuata, NaRALF occurs as a single-copy gene and is highly expressed in roots and petioles. Silencing the NaRALF transcript by transforming N. attenuata with an inverted-repeat construct generated plants (irRALF) with normal wild-type (WT) above-ground parts, but with roots that grew longer and produced trichoblasts that developed into abnormal root hairs. Most trichoblasts produced a localized 'bulge' without commencing root hair tip growth; fewer trichoblasts grew, but were only 10% as long as those of WT plants. The root hair phenotype was associated with slowed apoplastic pH oscillations, increased pH at the tips of trichoblasts and decreased accumulation of reactive oxygen species in the root hair initiation zone. The root hair growth phenotype was partially restored when irRALF lines were grown in a low-pH-buffered medium, and reproduced in WT plants grown in a high-pH-buffered medium. When irRALF plants were grown in pH 5.6, 6.7 and 8.1 soils together with WT plants in glasshouse experiments, they were out-competed by WT plants in basic, but not acidic, soils. When WT and irRALF lines were planted into the basic soils of the native habitat of N. attenuata in the Great Basin Desert, irRALF plants had smaller leaves, shorter stalks, and produced fewer flowers and seed capsules than did WT plants. We conclude that NaRALF is required for regulating root hair extracellular pH, the transition from root hair initiation to tip growth and plant growth in basic soils.

  17. Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. I. Large-Scale Changes in the Accumulation of Growth- and Defense-Related Plant mRNAs1

    PubMed Central

    Hermsmeier, Dieter; Schittko, Ursula; Baldwin, Ian T.

    2001-01-01

    Plants respond to herbivore attack with a dramatic functional reorganization that involves the activation of direct and indirect defenses and tolerance, which in turn make large demands on primary metabolism. Here we provide the first characterization of the transcriptional reorganization that occurs after insect attack in a model plant-herbivore system: Nicotiana attenuata Torr. ex Wats.-Manduca sexta. We used mRNA differential display to characterize one-twentieth of the insect-responsive transcriptome of N. attenuata and verified differential expression for 27 cDNAs. Northern analyses were used to study the effects of folivory and exposure to airborne methyl jasmonate and for kinetic analyses throughout a 16-h- light/8-h-dark cycle. Sequence similarity searches allowed putative functions to be assigned to 15 transcripts. Genes were related to photosynthesis, electron transport, cytoskeleton, carbon and nitrogen metabolism, signaling, and a group responding to stress, wounding, or invasion of pathogens. Overall, transcripts involved in photosynthesis were strongly down-regulated, whereas those responding to stress, wounding, and pathogens and involved in shifting carbon and nitrogen to defense were strongly up-regulated. The majority of transcripts responded similarly to airborne methyl jasmonate and folivory, and had tissue- and diurnal-specific patterns of expression. Transcripts encoding Thr deaminase (TD) and a putative retrotransposon were absent in control plants, but were strongly induced after herbivory. Full-length sequences were obtained for TD and the pathogen-inducible α-dioxygenase, PIOX. Effects of abiotic and biotic stimuli were investigated for transcripts encoding TD, importin α, PIOX, and a GAL83-like kinase cofactor. PMID:11161026

  18. Cytokinin concentrations and CHASE-DOMAIN CONTAINING HIS KINASE 2 (NaCHK2)- and NaCHK3-mediated perception modulate herbivory-induced defense signaling and defenses in Nicotiana attenuata.

    PubMed

    Schäfer, Martin; Meza-Canales, Ivan D; Brütting, Christoph; Baldwin, Ian T; Meldau, Stefan

    2015-08-01

    Herbivore attack elicits changes in cytokinins (CKs), but how these changes influence defense signaling remains poorly described. We investigated the influence of the CK pathway on the well-described inducible defense pathways of Nicotiana attenuata in response to wounding with and without elicitors from the specialist herbivore Manduca sexta. CK pathway manipulation often suffers from substantial side effects on plant growth and development. We therefore used multiple manipulation tools including spray application of CKs, chemically-inducible expression of the CK biosynthesis enzyme isopentenyltransferase, and transient and constitutive RNAi-mediated gene silencing of CK receptors to resolve the function of CKs in plant defense. The results demonstrated that CK concentrations in leaves and perception through CHASE-DOMAIN CONTAINING HIS KINASE 2 (NaCHK2) and NaCHK3 were important for the accumulation of jasmonic acid (JA) and phenolamides and proteinase inhibitor activity. By contrast, the CK pathway did not promote the accumulation of the active JA-isoleucine conjugate and negatively regulated the release of specific green leaf volatile esters. Interestingly, CK signaling also promotes the systemic phenolamide accumulation. We conclude that the CK pathway is an important regulator of herbivory-inducible defense signaling and chemistry, which expands its reported participation in adjusting a plant's physiology to abiotic and biotic stress responses. PMID:25919325

  19. In wild tobacco, Nicotiana attenuata, variation among bacterial communities of isogenic plants is mainly shaped by the local soil microbiota independently of the plants' capacity to produce jasmonic acid.

    PubMed

    Santhanam, Rakesh; Baldwin, Ian T; Groten, Karin

    2015-01-01

    The phytohormone jasmonic acid (JA) plays a central role in defense against necrotrophic pathogens and herbivores in Nicotiana attenuata. Recently Santhanam et al.(1) showed that JA does not have a major role in shaping the root- and shoot associated bacterial communities, though a few taxa differed among control (empty vector, EV) plants and plants impaired in their capacity to produce JA (irAOC). In this addendum, we provide additional data showing that the composition of the plant bacterial communities is mainly shaped by tissue type. The qualitative data analysis revealed that at the order level, 5 bacterial OTUs formed a core community found in all tissues irrespective of genotypes, while 9 OTUs were different among roots and shoots. The heterogeneity among individual plants was high masking the potential genotype effect on bacterial communities. Using a culture-dependent approach, 3 of 18 bacterial taxa retrieved either only from one of the genotypes or from both had a growth promoting effect on EV and irAOC seedlings. The data suggest that the local soil niche in which the roots grows is a major driver of the variability in root bacterial communities recruited by different individuals, and the plant growth-promoting effects of some taxa are independent of the genotype. PMID:26478769

  20. Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata: V. Microarray Analysis and Further Characterization of Large-Scale Changes in Herbivore-Induced mRNAs1

    PubMed Central

    Hui, Dequan; Iqbal, Javeed; Lehmann, Katja; Gase, Klaus; Saluz, Hans Peter; Baldwin, Ian T.

    2003-01-01

    We extend our analysis of the transcriptional reorganization that occurs when the native tobacco, Nicotiana attenuata, is attacked by Manduca sexta larvae by cloning 115 transcripts by mRNA differential display reverse transcription-polymerase chain reaction and subtractive hybridization using magnetic beads (SHMB) from the M. sexta-responsive transcriptome. These transcripts were spotted as cDNA with eight others, previously confirmed to be differentially regulated by northern analysis on glass slide microarrays, and hybridized with Cy3- and Cy5-labeled probes derived from plants after 2, 6, 12, and 24 h of continuous attack. Microarray analysis proved to be a powerful means of verifying differential expression; 73 of the cloned genes (63%) were differentially regulated (in equal proportions from differential display reverse transcription-polymerase chain reaction and SHMB procedures), and of these, 24 (32%) had similarity to known genes or putative proteins (more from SHMB). The analysis provided insights into the signaling and transcriptional basis of direct and indirect defenses used against herbivores, suggesting simultaneous activation of salicylic acid-, ethylene-, cytokinin-, WRKY-, MYB-, and oxylipin-signaling pathways and implicating terpenoid-, pathogen-, and cell wall-related transcripts in defense responses. These defense responses require resources that could be made available by decreases in four photosynthetic-related transcripts, increases in transcripts associated with protein and nucleotide turnover, and increases in transcripts associated with carbohydrate metabolism. This putative up-regulation of defense-associated and down-regulation of growth-associated transcripts occur against a backdrop of altered transcripts for RNA-binding proteins, putative ATP/ADP translocators, chaperonins, histones, and water channel proteins, responses consistent with a major metabolic reconfiguration that underscores the complexity of response to herbivore attack

  1. Nicotiana attenuata SIPK, WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway1[W][OA

    PubMed Central

    Kallenbach, Mario; Alagna, Fiammetta; Baldwin, Ian Thomas; Bonaventure, Gustavo

    2010-01-01

    Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants. PMID:19897603

  2. RuBPCase activase mediates growth-defense tradeoffs: Silencing RCA redirects JA flux from JA-Ile to MeJA to attenuate induced defense responses in Nicotiana attenuata

    PubMed Central

    Mitra, Sirsha; Baldwin, Ian T.

    2014-01-01

    Summary RuBPCase activase (RCA), an abundant photosynthetic protein is strongly down-regulated in response to Manduca sexta’s oral secretion (OS) in Nicotiana attenuata. RCA-silenced plants are impaired not only in photosynthetic capacity and growth, but also in jasmonic acid (JA)-isoleucine (Ile) signaling, and herbivore resistance mediated by JA-Ile dependent defense traits. These responses are consistent with a resource-based growth-defense trade-off. Since JA+Ile-supplementation of OS restored WT levels of JA-Ile, defenses and resistance to M. sexta, but OS supplemented individually with JA- or Ile did not, the JA-Ile deficiency of RCA-silenced plants could not be attributed to lower JA or Ile pools or JAR4/6 conjugating activity. Similar levels of JA-Ile derivatives after OS elicitation indicated unaltered JA-Ile turnover and lower levels of other JA-conjugates ruled out competition from other conjugation reactions. RCA-silenced plants accumulated more methyl jasmonate (MeJA) after OS elicitation, which corresponded with increased jasmonate methyltransferase (JMT) activity. RCA-silencing phenocopies JMT over-expression, wherein elevated JMT activity redirects OS-elicited JA flux towards inactive MeJA, creating a JA sink which depletes JA-Ile and its associated defense responses. Hence RCA plays an additional non-photosynthetic role in attenuating JA-mediated defenses and their associated costs potentially allowing plants to anticipate resource-based constraints on growth before they actually occur. PMID:24491116

  3. Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphigidae) and Its Natural Host Nicotiana attenuata. VIII. An Unbiased GCxGC-ToFMS Analysis of the Plant's Elicited Volatile Emissions[W][OA

    PubMed Central

    Gaquerel, Emmanuel; Weinhold, Alexander; Baldwin, Ian T.

    2009-01-01

    Treating wounds in Nicotiana attenuata leaves with Manduca sexta oral secretions (W+OS) mimics most changes elicited by M. sexta herbivory, but an unbiased analysis of the effect of the different OS constituents on volatile emissions is lacking. We used two-dimensional gas chromatography/time-of-flight (GCxGC-ToF) mass spectrometry combined with multivariate statistics to parse volatiles into regulatory patterns. Volatiles released by wounding alone and by the alkalinity of OS were assessed by applying a buffer known to mimic the pH-mediated changes of OS elicitation (pectin methyl esterase activation and methanol release). The activities of fatty acid amino acid conjugates, well-known elicitors of antiherbivore defenses, and of 2-hydroxyoctadecatrienoic acid, a newly discovered signal in OS, were determined. Approximately 400 analytes were detected after deconvolution and alignment of GCxGC data; 35 volatiles were significantly regulated upon W+OS. Two-thirds of these were specifically regulated by OS, being either amplified (most terpenoids and certain hexenylesters) or strongly repressed (many short-chain alcohols and some aromatic and hexenylester derivatives). Fatty acid amino acid conjugates played a central role in this pattern of regulation, since they induced the emission of half of OS-elicited volatiles and inhibited the production of almost all OS-repressed volatiles; 2-hydroxyoctadecatrienoic acid influenced emission of trans-α-bergamotene, while other unknown OS constituents amplified hexenylester production. We conclude that the complex bouquet of herbivory-elicited volatiles results from the complex modulations of the wound response by diverse cues found in OS. This work also underscores the value of ultra-high-resolution GCxGC-ToF analysis combined with the nontargeted mining of the resulting data. PMID:19136568

  4. A new steroidal saponin from Agave attenuata.

    PubMed

    Mendes, Tatiana Paz; Silva, Graziela de Medeiros; da Silva, Bernadete Pereira; Parente, José Paz

    2004-04-01

    A new steroidal saponin was isolated from the leaves of Agave attenuata. Its structure was established as (3beta,beta,25S)-spirostan-3-yl O-beta-D-glucopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 2)-O-[beta-D-glucopyranosyl-(1 --> 3)]-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranoside. The structural identification was performed using detailed analyses of 1H- and 13C-NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR, and COLOC) and chemical conversions. The hemolytic activity of the steroidal saponin was evaluated using an in vitro assay.

  5. Phytochemical and biological studies of Agave attenuata.

    PubMed

    Rizwan, Komal; Zubair, Muhammad; Rasool, Nasir; Riaz, Muhammad; Zia-Ul-Haq, Muhammad; de Feo, Vincenzo

    2012-01-01

    The present study was conducted to examine various biological activities of a methanol extract of Agave attenuata leaves. GC-MS analysis of the n-hexane fraction from the extract revealed the presence of 31 compounds, with mono-2-ethylhexyl phthalate (11.37%), 1,2-benzenedicarboxylic acid (6.33%), n-docosane (6.30%) and eicosane (6.02%) as the major components. The leaves contained appreciable levels of total phenolic contents (10.541-39.35 GAE, mg/100 g) and total flavonoid contents (43.35-304.8 CE, mg/100 g). The extract and some of its fractions showed moderate antimicrobial effects. Leaves extract and fractions also exhibited a good antioxidant potential when measured by DPPH radical scavenging activity and inhibition of lipid peroxidation assays. The hemolytic effect of the plant was found to be in a range of 1.01%-2.64%. From the present study it is concluded that this plant could be used as a source of natural antioxidants and functional food nutraceutical applications. PMID:22754375

  6. Phytochemical and biological studies of Agave attenuata.

    PubMed

    Rizwan, Komal; Zubair, Muhammad; Rasool, Nasir; Riaz, Muhammad; Zia-Ul-Haq, Muhammad; de Feo, Vincenzo

    2012-01-01

    The present study was conducted to examine various biological activities of a methanol extract of Agave attenuata leaves. GC-MS analysis of the n-hexane fraction from the extract revealed the presence of 31 compounds, with mono-2-ethylhexyl phthalate (11.37%), 1,2-benzenedicarboxylic acid (6.33%), n-docosane (6.30%) and eicosane (6.02%) as the major components. The leaves contained appreciable levels of total phenolic contents (10.541-39.35 GAE, mg/100 g) and total flavonoid contents (43.35-304.8 CE, mg/100 g). The extract and some of its fractions showed moderate antimicrobial effects. Leaves extract and fractions also exhibited a good antioxidant potential when measured by DPPH radical scavenging activity and inhibition of lipid peroxidation assays. The hemolytic effect of the plant was found to be in a range of 1.01%-2.64%. From the present study it is concluded that this plant could be used as a source of natural antioxidants and functional food nutraceutical applications.

  7. Phytochemical and Biological Studies of Agave attenuata

    PubMed Central

    Rizwan, Komal; Zubair, Muhammad; Rasool, Nasir; Riaz, Muhammad; Zia-Ul-Haq, Muhammad; de Feo, Vincenzo

    2012-01-01

    The present study was conducted to examine various biological activities of a methanol extract of Agave attenuata leaves. GC-MS analysis of the n-hexane fraction from the extract revealed the presence of 31 compounds, with mono-2-ethylhexyl phthalate (11.37%), 1,2-benzenedicarboxylic acid (6.33%), n-docosane (6.30%) and eicosane (6.02%) as the major components. The leaves contained appreciable levels of total phenolic contents (10.541–39.35 GAE, mg/100 g) and total flavonoid contents (43.35–304.8 CE, mg/100 g). The extract and some of its fractions showed moderate antimicrobial effects. Leaves extract and fractions also exhibited a good antioxidant potential when measured by DPPH radical scavenging activity and inhibition of lipid peroxidation assays. The hemolytic effect of the plant was found to be in a range of 1.01%–2.64%. From the present study it is concluded that this plant could be used as a source of natural antioxidants and functional food nutraceutical applications. PMID:22754375

  8. Cutaneous Phaeohyphomycosis Caused by Exophiala attenuata in a Domestic Cat.

    PubMed

    Overy, David P; Martin, Chelsea; Muckle, Anne; Lund, Lorraine; Wood, Jill; Hanna, Paul

    2015-10-01

    A 7-year-old female-spayed, domestic short-haired cat was presented to her veterinarian with a mass on the hind paw. Histopathologic examination of a tissue biopsy revealed nodular pyogranulomatous panniculitis with intralesional pigmented fungal hyphae. A dematiaceous fungal isolate was isolated with a micromorphological phenotype consistent with the anamorphic genus Exophiala: budding cells, torulose mycelium and annellidic conidiogenesis from simple conidiophores consisting of terminal and lateral cells that tapered to a short beak at the apex. Sequence homology of the internal transcribed spacer region of the rDNA gene confirmed the identification of the isolate as Exophiala attenuata. Reported here is the first confirmed case of feline phaeohyphomycosis caused by E. attenuata in North America. Similar to historical cases of feline phaeohyphomycosis caused by Exophiala spp., there was no history or postmortem evidence to suggest the patient was in an immunocompromised state (e.g., suffering from FeLV or FIV). Although aggressive surgical excision of local lesions is recommended prior to drug treatment when dealing with subcutaneous phaeohyphomycosis, surgery followed by itraconazole treatment did not resolve the E. attenuata infection in this cat. PMID:26088340

  9. Reference genomes and transcriptomes of Nicotiana sylvestris and Nicotiana tomentosiformis

    PubMed Central

    2013-01-01

    Background Nicotiana sylvestris and Nicotiana tomentosiformis are members of the Solanaceae family that includes tomato, potato, eggplant and pepper. These two Nicotiana species originate from South America and exhibit different alkaloid and diterpenoid production. N. sylvestris is cultivated largely as an ornamental plant and it has been used as a diploid model system for studies of terpenoid production, plastid engineering, and resistance to biotic and abiotic stress. N. sylvestris and N. tomentosiformis are considered to be modern descendants of the maternal and paternal donors that formed Nicotiana tabacum about 200,000 years ago through interspecific hybridization. Here we report the first genome-wide analysis of these two Nicotiana species. Results Draft genomes of N. sylvestris and N. tomentosiformis were assembled to 82.9% and 71.6% of their expected size respectively, with N50 sizes of about 80 kb. The repeat content was 72-75%, with a higher proportion of retrotransposons and copia-like long terminal repeats in N. tomentosiformis. The transcriptome assemblies showed that 44,000-53,000 transcripts were expressed in the roots, leaves or flowers. The key genes involved in terpenoid metabolism, alkaloid metabolism and heavy metal transport showed differential expression in the leaves, roots and flowers of N. sylvestris and N. tomentosiformis. Conclusions The reference genomes of N. sylvestris and N. tomentosiformis represent a significant contribution to the SOL100 initiative because, as members of the Nicotiana genus of Solanaceae, they strengthen the value of the already existing resources by providing additional comparative information, thereby helping to improve our understanding of plant metabolism and evolution. PMID:23773524

  10. Evolution of proteinase inhibitor defenses in North American allopolyploid species of Nicotiana.

    PubMed

    Wu, Jianqiang; Hettenhausen, Christian; Baldwin, Ian T

    2006-09-01

    We studied the jasmonate (JA)-elicited trypsin-proteinase inhibitor (TPI) anti-herbivore defense system in North American Nicotiana to understand how complex polygenetic traits evolve after allopolyploidy speciation. N. quadrivalvis (Nq) and N. clevelandii (Nc) are allotetraploid descendant species of the ancestors of the diploid species N. attenuata (Na) and N. obtusifolia (No). From cDNA, intron and promoter sequence analyses, and Southern blotting, we deduced that only the maternally derived No TPI genes were retained in the tetraploid genomes (Nq, Nc), whereas the sequences of the paternal Na ancestor were deleted. The number of TPI repeats in different Nicotiana taxa was independent of phylogenetic associations. In Na, TPI activity and mRNA transcript accumulation as well as JA levels increased dramatically above wound-induced levels when the oral secretions (OS) from Manduca sexta larvae were introduced into wounds. This OS-mediated amplification of defense signaling and downstream response was also found in the tetraploid genomes but was absent from No; in No, OS treatment suppresses TPI mRNA accumulation and activity and does not increase JA accumulation. Hence, the tetraploids retained components of Na's signaling system, but lost Na's TPI genes and used No's TPI genes to retain a functional TPI defense system, underscoring the genomic flexibility that enables complex polygenic traits to be retained in allopolyploid species.

  11. A new bioactive steroidal saponin from Agave attenuata.

    PubMed

    da Silva, Bernadete P; de Sousa, Allyne C; Silva, Graziela M; Mendes, Tatiana P; Parente, José P

    2002-01-01

    A new steroidal saponin was isolated from the leaves of Agave attenuata Salm-Dyck. Its structure was established as (3beta,5beta,22alpha,25S)-26-(beta-D-glucopyranosyloxy)-22-methoxyfurostan-3-yl O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->3)]-beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside. The structural identification was performed using detailed analyses of 1H and 13C NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR and COLOC) and chemical conversions. The haemolytic potential of the steroidal saponin was evaluated and the anti-inflammatory activity was performed using the capillary permeability assay.

  12. Leaf surface chemicals fromNicotiana affecting germination ofPeronospora tabacina (adam) sporangia.

    PubMed

    Kennedy, B S; Nielsen, M T; Severson, R F; Sisson, V A; Stephenson, M K; Jackson, D M

    1992-09-01

    A bioassay was used to evaluate the effects of cuticular leaf components, isolated fromN. tabacum, N. glutinosa (accessions 24 and 24a), and 23other Nicotiana species, on germinationof P. tabacina (blue mold). The leaf surface compounds includedα- andβ-4,8,13,-duvatriene-l,3-diols (DVT-diols), (13-E)-labda-13-ene-8α-,15-diol (labdenediol), (12-Z)-labda-12,14-diene-8α-ol (cis-abienol), (13-R)-labda-8,14-diene-13-ol (manool), 2-hydroxymanool, a mixture of (13-R)-labda-14-ene-8α,13-diol (sclareol), and (13-S)-labda-14-ene-8α,13-diol (episclareol), and various glucose and/or sucrose ester isolates. The above in acetone were applied onto leaf disks of the blue moldsusceptibleN. tabacum cv. TI 1406, which was then inoculated with blue mold sporangia. Estimated IC50 values (inhibitory concentration) were 3.0μg/cm(2) forα-DVT-diol, 2.9μ/cm(2) forβ-DVT-diol, 0.4μg/cm(2) for labdenediol and 4.7μg/cm(2) for the sclareol mixture. Manool, 2-hydroxymanool, andcis-abienol at application rates up to 30μg/cm(2) had little or no effect on sporangium germination. Glucose and/or sucrose ester isolates from the cuticular leaf extracts of 23Nicotiana species and three different fractions fromN. bigelovii were also evaluated for antimicrobial activity at a concentration of 30μg/cm(2). Germination was inhibited by >20% when exposed to sugar esters isolated fromN. acuminata, N. benthamiana, N. attenuata, N. clevelandii, andN. miersii, and accessions 10 and 12 ofN. bigelovii. These results imply that a number of compounds may influence resistance to blue mold in tobacco. PMID:24254279

  13. Alternative life cycle strategies and colonization of young anurans by Gorgoderina attenuata in Nebraska.

    PubMed

    Bolek, Matthew G; Snyder, Scott D; Janovy, John

    2009-06-01

    Studies on life cycles and epizootiology of North American frog bladder flukes indicate that adult frogs become infected predominantly by ingesting tadpoles or other frogs that serve as second intermediate hosts for gorgoderid metacercariae. Other studies have indicated that newly metamorphosed frogs are rarely infected with these parasites because they are gape-limited predators that cannot feed on large intermediate hosts such as tadpoles and other frogs. We examined the role of potential intermediate hosts in the recruitment of the frog bladder fluke, Gorgoderina attenuata, to metamorphosed northern leopard frogs, Woodhouse's toads, and bullfrogs from western Nebraska. We completed the life cycle of G. attenuata in the laboratory in 3 anuran species by experimentally infecting a variety of hosts. In addition, we generated and compared DNA sequence data from life cycle stages collected from a variety of naturally infected hosts. Our field and laboratory data indicate that in Nebraska G. attenuata has a truncated, 2-host life cycle that includes fingernail clams and anurans. Cercariae are ingested directly by tadpoles; unencysted juvenile worms then develop in the kidneys of tadpoles before moving to, and maturing in, the urinary bladder when tadpoles metamorphose. Additionally, G. attenuata can infect metamorphosed leopard frogs, bullfrogs, and toads when metacercariae in damselfly second intermediate hosts are ingested. These worms can also infect adult bullfrogs when they feed on other infected anurans possessing worms in their kidneys. Comparison of our material to published accounts of G. attenuata morphology and life cycles in Massachusetts suggests that previous work may have inadvertently involved 2 different species of gorgoderids. Our comparative approach to life cycle studies in different anuran life stages and multiple species of hosts suggests that tadpoles and metamorphosed anurans have favored alternative life cycle strategies in this trematode

  14. Alternative life cycle strategies and colonization of young anurans by Gorgoderina attenuata in Nebraska.

    PubMed

    Bolek, Matthew G; Snyder, Scott D; Janovy, John

    2009-06-01

    Studies on life cycles and epizootiology of North American frog bladder flukes indicate that adult frogs become infected predominantly by ingesting tadpoles or other frogs that serve as second intermediate hosts for gorgoderid metacercariae. Other studies have indicated that newly metamorphosed frogs are rarely infected with these parasites because they are gape-limited predators that cannot feed on large intermediate hosts such as tadpoles and other frogs. We examined the role of potential intermediate hosts in the recruitment of the frog bladder fluke, Gorgoderina attenuata, to metamorphosed northern leopard frogs, Woodhouse's toads, and bullfrogs from western Nebraska. We completed the life cycle of G. attenuata in the laboratory in 3 anuran species by experimentally infecting a variety of hosts. In addition, we generated and compared DNA sequence data from life cycle stages collected from a variety of naturally infected hosts. Our field and laboratory data indicate that in Nebraska G. attenuata has a truncated, 2-host life cycle that includes fingernail clams and anurans. Cercariae are ingested directly by tadpoles; unencysted juvenile worms then develop in the kidneys of tadpoles before moving to, and maturing in, the urinary bladder when tadpoles metamorphose. Additionally, G. attenuata can infect metamorphosed leopard frogs, bullfrogs, and toads when metacercariae in damselfly second intermediate hosts are ingested. These worms can also infect adult bullfrogs when they feed on other infected anurans possessing worms in their kidneys. Comparison of our material to published accounts of G. attenuata morphology and life cycles in Massachusetts suggests that previous work may have inadvertently involved 2 different species of gorgoderids. Our comparative approach to life cycle studies in different anuran life stages and multiple species of hosts suggests that tadpoles and metamorphosed anurans have favored alternative life cycle strategies in this trematode.

  15. Ecological divergence and evolutionary transition of resprouting types in Banksia attenuata

    PubMed Central

    He, Tianhua

    2014-01-01

    Resprouting is a key functional trait that allows plants to survive diverse disturbances. The fitness benefits associated with resprouting include a rapid return to adult growth, early flowering, and setting seed. The resprouting responses observed following fire are varied, as are the ecological outcomes. Understanding the ecological divergence and evolutionary pathways of different resprouting types and how the environment and genetics interact to drive such morphological evolution represents an important, but under-studied, topic. In the present study, microsatellite markers and microevolutionary approaches were used to better understand: (1) whether genetic differentiation is related to morphological divergence among resprouting types and if so, whether there are any specific genetic variations associated with morphological divergence and (2) the evolutionary pathway of the transitions between two resprouting types in Banksia attenuata (epicormic resprouting from aerial stems or branch; resprouting from a underground lignotuber). The results revealed an association between population genetic differentiation and the morphological divergence of postfire resprouting types in B. attenuata. A microsatellite allele has been shown to be associated with epicormic populations. Approximate Bayesian Computation analysis revealed a likely evolutionary transition from epicormic to lignotuberous resprouting in B. attenuata. It is concluded that the postfire resprouting type in B. attenuata is likely determined by the fire's characteristics. The differentiated expression of postfire resprouting types in different environments is likely a consequence of local genetic adaptation. The capacity to shift the postfire resprouting type to adapt to diverse fire regimes is most likely the key factor explaining why B. attenuata is the most widespread member of the Banksia genus. PMID:25473470

  16. Emerging antibody products and Nicotiana manufacturing.

    PubMed

    Whaley, Kevin J; Hiatt, Andrew; Zeitlin, Larry

    2011-03-01

    Antibody based products are not widely available to address multiple global health challenges due to high costs, limited manufacturing capacity, and long manufacturing lead times. Nicotiana-based manufacturing of antibody products may now begin to address these challenges as a result of revolutionary advances in transient expression and altered glycosylation pathways. This review provides examples of emerging antibody-based products (mucosal and systemic) that could be competitive and commercially viable when the attributes of Nicotiana-based manufacturing (large scale, versatile, rapid, low cost) are utilized.

  17. [Sighting of Stenella attenuata, the spotted dolphin, in Culebra Bay, Costa Rica, 1999-2000].

    PubMed

    Rodríguez Sáenz, Karina; Rodríguez-Fonseca, Javier

    2004-12-01

    Parallel to a zooplankton study (1999-2000) observations were made (from an inflatable boat), on the presence of dolphins along a transect (-8 km long) on the axis of Culebra Bay (24 km2), Gulf of Papagayo, Pacific coast of Costa Rica. Dolphins were found during 20 of the 31 boat surveys conducted. The only species of cetacean found in the bay was Stenella attenuata, the spotted dolphin. These sightings were more frequent during the rainy season, particularly during the month of May of both years. The presence of S. attenuata in Culebra Bay might be associated to the abundances of fish and mollusks (their presumed prey: for example, squids), as evidenced by fishery statistics available for this zone of the Pacific coast of Costa Rica. PMID:17465137

  18. Ulcer healing potential of ethanolic extract of Caralluma attenuata on experimental diabetic rats

    PubMed Central

    Garg, Sunil; Srivastava, Sajal; Singh, Kisanpal; Sharma, Alok; Garg, Kavita

    2016-01-01

    Introduction: Available data indicated that diabetes mellitus (DM) increases the vulnerability of the gastric ulcers and the need of the hour is to develop effective agents to treat ulcer with diabetes for better patient compliance and cost effectiveness. The ulcer-healing properties of ethanolic extract of Caralluma attenuata (CAEt) against both chemically- and physically induced gastric ulcers in experimental rats are recently studied. Aim: To assess the ulcer healing potential of Ethanolic Extract of Caralluma attenuata on Experimental Diabetic Rats. Material and Methods: The current study aimed to evaluate ulcer healing properties of CAEt on the aspirin induced gastric ulcer in rats with streptozotocin induced DM. The hypothesis is based on the fact that DM results in compromising the mucosal defensive factors associated with delay in gastric ulcer healing, and if these changes can be corrected by using agents known for their antidiabetic and antiulcer properties. Experimental albino rats were divided into six groups. Except for Group I, other groups contained streptozotocin-induced diabetic rats. Group I (normal control) and Group II (diabetic control) were administered vehicle, Groups III and IV (diabetic experimental) were administered CAEt in dose of 100 mg/kg and 250 mg/kg, respectively, and Groups V and VI (positive controls) were respectively administered oral standard drugs omeprazole, 20 mg/kg, and tolbutamide 10 mg/kg. Result: The results confirmed that the CAEt significantly decreases the ulcer index (P < 0.05) in the aspirin-induced gastric ulcers and also significantly exhibit antioxidant and glucose lowering activity in the diabetic ulcer rats. The study showed that C. attenuata has the potential to be used as an antiulcer agent in experimental diabetic rats.

  19. Ulcer healing potential of ethanolic extract of Caralluma attenuata on experimental diabetic rats

    PubMed Central

    Garg, Sunil; Srivastava, Sajal; Singh, Kisanpal; Sharma, Alok; Garg, Kavita

    2016-01-01

    Introduction: Available data indicated that diabetes mellitus (DM) increases the vulnerability of the gastric ulcers and the need of the hour is to develop effective agents to treat ulcer with diabetes for better patient compliance and cost effectiveness. The ulcer-healing properties of ethanolic extract of Caralluma attenuata (CAEt) against both chemically- and physically induced gastric ulcers in experimental rats are recently studied. Aim: To assess the ulcer healing potential of Ethanolic Extract of Caralluma attenuata on Experimental Diabetic Rats. Material and Methods: The current study aimed to evaluate ulcer healing properties of CAEt on the aspirin induced gastric ulcer in rats with streptozotocin induced DM. The hypothesis is based on the fact that DM results in compromising the mucosal defensive factors associated with delay in gastric ulcer healing, and if these changes can be corrected by using agents known for their antidiabetic and antiulcer properties. Experimental albino rats were divided into six groups. Except for Group I, other groups contained streptozotocin-induced diabetic rats. Group I (normal control) and Group II (diabetic control) were administered vehicle, Groups III and IV (diabetic experimental) were administered CAEt in dose of 100 mg/kg and 250 mg/kg, respectively, and Groups V and VI (positive controls) were respectively administered oral standard drugs omeprazole, 20 mg/kg, and tolbutamide 10 mg/kg. Result: The results confirmed that the CAEt significantly decreases the ulcer index (P < 0.05) in the aspirin-induced gastric ulcers and also significantly exhibit antioxidant and glucose lowering activity in the diabetic ulcer rats. The study showed that C. attenuata has the potential to be used as an antiulcer agent in experimental diabetic rats. PMID:27621520

  20. Development of the skull of the pantropical spotted dolphin (Stenella attenuata).

    PubMed

    Moran, Meghan M; Nummela, Sirpa; Thewissen, J G M

    2011-10-01

    We describe the bony and cartilaginous structures of five fetal skulls of Stenella attenuata (pantropical spotted dolphin) specimens. The specimens represent early fetal life as suggested by the presence of rostral tactile hairs and the beginnings of skin pigmentation. These specimens exhibit the developmental order of ossification of the intramembranous and endochondral elements of the cranium as well as the functional and morphological development of specific cetacean anatomical adaptations. Detailed observations are presented on telescoping, nasal anatomy, and middle ear anatomy. The development of the middle ear ossicles, ectotympanic bone, and median nasal cartilage is of interest because in the adult these structures are morphologically different from those in land mammals. We follow specific cetacean morphological characteristics through fetal development to provide insight into the form and function of the cetacean body plan. Combining these data with fossil evidence, it is possible to overlie ontogenetic patterns and discern evolutionary patterns of the cetacean skull. PMID:21901843

  1. Source parameter estimates of echolocation clicks from wild pygmy killer whales (Feresa attenuata) (L)

    NASA Astrophysics Data System (ADS)

    Madsen, P. T.; Kerr, I.; Payne, R.

    2004-10-01

    Pods of the little known pygmy killer whale (Feresa attenuata) in the northern Indian Ocean were recorded with a vertical hydrophone array connected to a digital recorder sampling at 320 kHz. Recorded clicks were directional, short (25 μs) transients with estimated source levels between 197 and 223 dB re. 1 μPa (pp). Spectra of clicks recorded close to or on the acoustic axis were bimodal with peak frequencies between 45 and 117 kHz, and with centroid frequencies between 70 and 85 kHz. The clicks share characteristics of echolocation clicks from similar sized, whistling delphinids, and have properties suited for the detection and classification of prey targeted by this odontocete. .

  2. Glutamine Synthetase of Nicotiana plumbaginifolia1

    PubMed Central

    Tingey, Scott V.; Coruzzi, Gloria M.

    1987-01-01

    We have characterized the distinct forms of glutamine synthetase (GS) which are present in leaves and roots of Nicotiana plumbaginifolia. Mature leaves contain a single GS polypeptide (44 kilodaltons in size) which is localized to the stroma of intact chloroplasts. In contrast, the GS polypeptide in roots is distinct in size (38 kilodaltons) and charge. A lectin stain of leaf soluble protein indicates that the size difference of these mature GS polypeptides is not the result of posttranslational glycosylation. cDNA clones encoding a GS mRNA of N. plumbaginifolia were characterized and used as molecular probes to examine GS transcripts in leaves and roots. GS mRNA hybrid-selected from leaves or roots translated in vitro into distinct GS primary translation products (49 or 38 kilodaltons). The 49 kilodalton GS primary translation product, specific to leaf poly(A)RNA is proposed to be a precursor to the mature 44 kilodalton chloroplast stromal GS polypeptide. The 38 kilodalton GS primary translation product encoded by root GS mRNA, corresponds in size to the polypeptide encoded by the GS cDNA clones characterized. Southern blot analysis of nuclear DNA indicates that there are several different genomic fragments encoding GS in N. plumbaginifolia. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:16665445

  3. Application of the Hydra attenuata assay for identifying developmental hazards among natural waters and wastewaters

    SciTech Connect

    Fu, L.J.; Staples, R.E.; Stahl, R.G. Jr. )

    1991-12-01

    This study concerns application of the Hydra attenuata assay to detect the developmental toxicity potential of various aqueous samples. First, the assay was modified for testing aqueous samples because water quality has a major impact on aquatic toxicity testing and the results thus obtained. Ranges of sample pH, salinity (conductivity), and hardness were examined for their adverse effects upon the hydra. Adult hydra were unaffected morphologically by pH 5.5-9.5, and the artificial embryo ( embryo') developed normally in a pH range of 6.25 to 8.25. For water hardness, the minimal affective concentration was 1000 mg/liter (as CaCO3) in adults and 625 mg/liter in the embryos; the NOAELs for these were 750 mg/liter in the adult and 250 mg/liter CaCO3 in the embryo. Salinity in excess of 5 ppt was lethal to adults and embryos, indicating the assay may not be applicable to marine or highly saline samples. Finally, grab samples were tested from rivers in Maryland, Pennsylvania, New Jersey, and Delaware, some of which are impacted by industrial and agricultural activities, as well as several samples of industrial wastewaters from one major facility. The assay functioned normally with these diverse samples and yielded results that can be used in assessing the potential developmental hazard of these materials.

  4. Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

    PubMed

    Jones, B N; Wang, C C; Dwulet, F E; Lehman, L D; Meuth, J L; Bogardt, R A; Gurd, F R

    1979-04-25

    The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea. PMID:454657

  5. Cranial variation in the pantropical spotted dolphin, Stenella attenuata , in the Pacific Ocean.

    PubMed

    Yao, Chiou-Ju; Yamada, Tadasu K; Chen, Yen-Jean; Chou, Lien-Siang

    2008-12-01

    Cranial variation in 129 pantropical spotted dolphins (Stenella attenuata) from the western and eastern tropical Pacific was investigated morphometrically. This study revealed significant sexual dimorphism in the dolphin skulls. Differences between the genders were mostly recorded in the rostrum and braincase, which are wider and more robust in males. This sexual variation was interpreted in terms of differences in behavioral and life-history strategies between genders. Geographical variation among populations of the eastern tropical Pacific (ETP), Japan, and Taiwan was also significant, with different sexual patterns. While no clear trend in geographical variation was detected in males, there was a positive relationship in females between morphological differences and geographical distance. That is, Japanese and Taiwanese female specimens were morphologically more similar than revealed by other pairwise comparisons between them and the ETP specimens. Moreover, two canonical discriminant functions further discriminated the three populations for males and females, respectively. The two functions showed that the rostrum and braincase are the principal elements in discrimination of geographical variation in males, while only the rostrum is the major discriminant morph of geographical variation in females. In addition, tests based on Mahalanobis distance-squares were conducted to assign six specimens from the western tropical Pacific to the above three populations to clarify the similarities among them. PMID:19267651

  6. On the development of Cetacean extremities: I. Hind limb rudimentation in the Spotted dolphin (Stenella attenuata).

    PubMed

    Sedmera, D; Misek, I; Klima, M

    1997-02-01

    The Cetacea are group of animals which have completely lost their hind limbs during the course of evolution as a result of their entirely aquatic mode of life. It is known, however, that during their embryonal period, the hind limb buds are temporarily present. The control mechanisms of this regression are not yet understood, and vestigial limbs can sometimes be found in adults. The aim of the present study is to describe the course of hind limb rudimentation during prenatal development of Stenella attenuata (Spotted dolphin) at tissue and cell levels and compare the results with other natural or experimentally induced amelias. Hind limb buds of dolphin embryos, CRL 10-30 mm, were examined histologically. Before total disappearance, they show histodifferentiation comparable with other mammals. Initially, they form the apical ectodermal ridge, which soon regresses. The mesenchyme undergoes the process of condensation to form anlagens of prospective skeletal elements. These condensations are surrounded by vascular plexuses. During the course of rudimentation, some mesenchymal cells die, while the others are incorporated into the body wall. Nerve ingrowth into rudimentary limb buds was also detected. The temporary presence of hind limb rudiments in cetacean embryos can be regarded as a good example of recapitulation of phylogenesis in ontogenesis. PMID:9143876

  7. Role of Crassicauda sp. in natural mortality of pantropical spotted dolphins Stenella attenuata: a reassessment.

    PubMed

    Balbuena, Juan Antonio; Simpkin, Andrew

    2014-02-01

    Evaluating the effect of parasites on population size is essential for designing management and conservation plans of wild animal populations. Although knowledge in this area is scarce in cetaceans, current evidence suggests that species of the nematode genus Crassicauda may play an important regulatory role in some populations. In the present study, a semiparametric regression technique was applied to a previously published dataset to re-examine the role of Crassicauda sp. in natural mortality of pantropical spotted dolphins Stenella attenuata. The resulting model indicated parasite-induced mortality at ages between 6.5 and 9 yr and at roughly 12 yr. The maximum mortality estimates obtained could represent 2 to 4% of natural mortality in dolphins 6 to 8 yr old. This estimate is substantially smaller than previously published values, but in contrast with previous research, our model provides clear statistical evidence for parasite-induced mortality because the bootstrapped 95% confidence intervals of the estimated mortality rates excluded the 0 value. We also evaluated, through simulations, how potential sampling biases of infected dolphins could overestimate parasite-induced mortality. Small differences in sampling selectivity between infected and uninfected animals could substantially reduce the mortality estimates. However, the simulated models also supported the notion of statistically significant mortality in juvenile dolphins. Given that dolphins older than 16 yr were poorly represented in the dataset, further research is needed to establish whether Crassicauda sp. causes meaningful mortality for population dynamics among adult individuals. PMID:24492057

  8. Application of the Hydra attenuata assay for identifying developmental hazards among natural waters and wastewaters.

    PubMed

    Fu, L J; Staples, R E; Stahl, R G

    1991-12-01

    This study concerns application of the Hydra attenuata assay to detect the developmental toxicity potential of various aqueous samples. First, the assay was modified for testing aqueous samples because water quality has a major impact on aquatic toxicity testing and the results thus obtained. Ranges of sample pH, salinity (conductivity), and hardness were examined for their adverse effects upon the hydra. Adult hydra were unaffected morphologically by pH 5.5-9.5, and the artificial embryo ("embryo") developed normally in a pH range of 6.25 to 8.25. For water hardness, the minimal affective concentration was 1000 mg/liter (as CaCO3) in adults and 625 mg/liter in the embryos; the NOAELs for these were 750 mg/liter in the adult and 250 mg/liter CaCO3 in the embryo. Salinity in excess of 5 ppt was lethal to adults and embryos, indicating the assay may not be applicable to marine or highly saline samples. Finally, grab samples were tested from rivers in Maryland, Pennsylvania, New Jersey, and Delaware, some of which are impacted by industrial and agricultural activities, as well as several samples of industrial wastewaters from one major facility. The assay functioned normally with these diverse samples and yielded results that can be used in assessing the potential developmental hazard of these materials.

  9. First record of Pantropical spotted dolphins Stenella attenuata in the Yellow Sea, China

    NASA Astrophysics Data System (ADS)

    Wu, Fuxing; Wang, Xianyan; Zhang, Qiuxia; Miao, Xing; Zhang, Ting; Zhu, Qian

    2015-07-01

    On October 1, 2009, sixteen dolphins were obtained from fishermen by incidental catching in the Yellow Sea, China. As the dolphins' skin color was ambiguous, morphological parameters were measured, and mitochondrial DNA cytochrome b (Cyt b) gene sequence was studied to identify the species. Morphological characteristics were consistent with Pantropical spotted dolphins, Stenella attenuata. Furthermore, a partial mitochondrial DNA cytochrome b (Cyt b) gene sequence as long as 328-bp was studied by extracting genomic DNA from the skins, and six haplotypes were detected in the sixteen dolphins. By comparing homologous sequences available in GenBank (www.ncbi.nlm.nih.gov), all the six haplotypes had maximal genetic similarity with Pantropical spotted dolphin. Eight species of cetacean (whales and dolphins) are now recognised in the Yellow Sea. To the best of our knowledge, this is the first record of Pantropical spotted dolphins from this region. Despite this species being listed as a Grade II National Key Protected Animal since 1988, little is known of its biology in Chinese waters. We recommend remedial research be undertaken to ensure appropriate management.

  10. Thylakoid formation from coiled lamellar bodies during carposporogenesis in Faucheocolax attenuata Setch. (Rhodophyta, Rhodymeniales).

    PubMed

    Delivopoulos, S G; Kugrens, P

    1985-04-01

    Chloroplast development during carposporogenesis in the parasitic red alga Faucheocolax attenuata Setch. was studied by electron microscopy. Proplastids are usually found in the peripheral cytoplasm of young carpospores and are characterized by the presence of portions of a peripheral thylakoid and coiled lamellar bodies that range in size up to 0.5 micron. One type of coiled lamellar body occurs in the peripheral region of the proplastid and is continuous with the peripheral thylakoid, while the other type is found in the central portion of the stroma. These coiled lamellae separate and expand, adding membranes to both thylakoid systems, thereby functioning as thylakoid-forming bodies. As each coiled lamella unravels, it forms an undulated double-membraned structure having the same width as a thylakoid. After substantial expansion, the developing thylakoids begin to straighten and assume a parallel orientation to each other, thus becoming mature thylakoids. Small coiled lamellae often persist in mature carpospore chloroplasts, and are utilized in additional thylakoid formation during carpospore germination.

  11. Phylogenetic fragrance patterns in Nicotiana sections Alatae and Suaveolentes.

    PubMed

    Raguso, Robert A; Schlumpberger, Boris O; Kaczorowski, Rainee L; Holtsford, Timothy P

    2006-09-01

    We analyzed floral volatiles from eight tobacco species (Nicotiana; Solanaceae) including newly discovered Brazilian taxa (Nicotiana mutabilis and "Rastroensis") in section Alatae. Eighty-four compounds were found, including mono- and sesquiterpenoids, nitrogenous compounds, benzenoid and aliphatic alcohols, aldehydes and esters. Floral scent from recent accessions of Nicotiana alata, Nicotiana bonariensis and Nicotiana langsdorffii differed from previously published data, suggesting intraspecific variation in scent composition at the level of biosynthetic class. Newly discovered taxa in Alatae, like their relatives, emit large amounts of 1,8-cineole and smaller amounts of monoterpenes on a nocturnal rhythm, constituting a chemical synapomorphy for this lineage. Fragrance data from three species of Nicotiana sect. Suaveolentes, the sister group of Alatae, (two Australian species: N. cavicola, N. ingulba; one African species: N. africana), were compared to previously reported data from their close relative, N. suaveolens. Like N. suaveolens, N. cavicola and N. ingulba emit fragrances dominated by benzenoids and phenylpropanoids, whereas the flowers of N. africana lacked a distinct floral scent and instead emitted only small amounts of an aliphatic methyl ester from foliage. Interestingly, this ester also is emitted from foliage of N. longiflora and N. plumbaginifolia (both in section Alatae s.l.), which share a common ancestor with N. africana. This result, combined with the synapomorphic pattern of 1,8 cineole emission in Alatae s.s., suggests that phylogenetic signal explains a major component of fragrance composition among tobacco species in sections Alatae and Suaveolentes. At the intraspecific level, interpopulational scent variation is widespread in sect. Alatae, and may reflect edaphic specialization, introgression, local pollinator shifts, genetic drift or artificial selection in cultivation. Further studies with genetically and geographically well

  12. Silicon delays tobacco Ringspot virus systemic symptoms in Nicotiana tabacum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soluble silicon (Si) provides protection to plants against a variety of abiotic and biotic stress. However, the role of Si in viral infections has been elusive. To investigate the role of Si in viral infections, hydroponic studies were conducted in Nicotiana tabacum with two pathogens: Tobacco rings...

  13. Toxicity of nonylphenol on the cnidarian Hydra attenuata and environmental risk assessment.

    PubMed

    Pachura-Bouchet, S; Blaise, C; Vasseur, P

    2006-08-01

    Alkylphenols and their derivatives, alkylphenol polyethoxylates (APEs), are synthetic chemicals of concern owing to their endocrine properties. Nonylphenol (NP) is a critical APE metabolite because of its recalcitrance to biodegradation, toxicity, and ability to bio-accumulate in aquatic organisms. Studies of NP effects in vertebrates demonstrated estrogenic disrupting properties in fish, birds, reptiles, and mammal cells in which NP displaces the natural estrogen from its receptor. Less is known on its toxicity toward invertebrates. Effects on reproduction have been reported, but toxicity on development has been poorly documented thus far. We investigated NP toxicity on survival and regeneration of the freshwater coelenterate Hydra attenuata. Hydra is known for its regenerative capacity and its sensitivity to chemical pollution. It has been used for over 20 years to screen for teratogenicity of chemicals (Johnson et al. (1982) Teratog Carcinog Mutagen 2:263-276). Our results showed that hydra appeared as one of the most sensitive species to acute and chronic toxicity of NP compared to several freshwater invertebrates. Regeneration was disrupted at NP concentrations lower than those affecting survival. Toxicity thresholds of NP for aquatic vertebrates and invertebrates are also reported and discussed in the context of environmental risk assessment and of water quality objectives recommended for surface waters in industrialized countries. NP levels have decreased during the last decade because of a voluntary agreement of surfactant producers and users. At present, concentrations of NP found in surface waters are far below 1 microg/L in Europe, but can reach several microg/L when wastewater treatment plant inefficiency occurs. PMID:16841324

  14. Jasmonoyl-l-isoleucine hydrolase 1 (JIH1) contributes to a termination of jasmonate signaling in N. attenuata

    PubMed Central

    Woldemariam, Melkamu G; Gális, Ivan; Baldwin, Ian T

    2014-01-01

    The jasmonate signaling pathway is essential for plant development, reproduction, and defense against herbivores and pathogens. When attacked by herbivores, plants elicit defense responses through the rapid accumulation of jasmonates. Although the transduction of the jasmonate burst into downstream responses has been largely resolved in the past decade, how the jasmonate burst is switched off remained unknown. Recently, two mechanisms that involve cytochrome p450-mediated hydroxylation/carboxylation and NaJIH1-mediated hydrolysis of JA-Ile were identified as major termination mechanisms of JA signaling. Due to a lack of hydrolysis, N. attenuata plants silenced in the expression of the JIH1 gene accumulated significantly more JA-Ile than did wild type plants and became more resistant to herbivore attack. Although less likely, additional functions of JIH1, such as contributing to the pool of free Ile and thereby increasing JA-Ile accumulation, remained untested. Here we show that increased isoleucine availability does not explain the observed phenotype in JIH1-deficient N. attenuata plants. PMID:24776843

  15. Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance

    PubMed Central

    Beseli, Aydin; Noar, Roslyn; Daub, Margaret E.

    2015-01-01

    The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species’ resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta

  16. Isolation of viable sperm cells from tobacco (Nicotiana tabacum).

    PubMed

    Cao, Y; Reece, A; Russell, S D

    1996-05-01

    Viable sperm cells of Nicotiana tabacum were isolated by the semi-vivo technique. After pollination, excised styles were floated, cut end immersed, in a solution of 15% sucrose with 0.01% boric acid and 0.03% Ca(NO3)2 at 27 degrees C in a growth chamber until pollen tubes emerged. After sperm cells were formed (at least 8 h after pollination) tubes were immersed in a 9% mannitol solution. In this solution, sperm cells are nearly ellipsoidal and retain viability for over 6 h.

  17. Genetic analysis of Phytophthora nicotianae populations from different hosts using microsatellite markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two hundred thirty-one isolates of P. nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa and Ruta) and other plant genera of lesser econo...

  18. A morphological and histological examination of the pan-tropical spotted dolphin (Stenella attenuata) and the spinner dolphin (Stenella longirostris) adrenal gland.

    PubMed

    Clark, L S; Cowan, D F; Pfeiffer, D C

    2008-04-01

    The morphology and histology of the cetacean adrenal gland are poorly understood. Therefore, this study examined 32 pairs of adrenal glands from 18 pan-tropical spotted dolphins (Stenella attenuata) and 14 spinner dolphins (Stenella longirostris). In both species, the cortex was pseudolobulated and contained a typical mammalian zonation. Medullary protrusions (0-3 per section) and a medullary band were identified in both species. For S. attenuata, no statistical differences were found in the cortex to medulla (CM) ratio or the percent cross-sectional area (PCA) of the adrenal glands compared with sex or sexual maturity. The mean CM ratio for S. attenuata was 2.34 and the PCA was 64.4% cortex, 29.4% medulla and 6.2%'other'. 'Other' indicates blood vessels, connective tissue and the gland capsule itself. For S. longirostris, there was no statistical difference in the CM ratio compared with sexual maturity. However, a statistical difference was found between the CM ratio and sex, suggesting sexual dimorphism (female CM ratio = 2.46 and males = 3.21). No statistical differences were found in the PCA of S. longirostris adrenal glands by sexual maturity. However, a statistical difference was found between the PCA by sex. Female S. longirostris adrenal glands consisted of 65.0% cortex, 27.3% medulla and 7.7% 'other', whereas male adrenal glands consisted of 71.7% cortex, 22.7% medulla and 5.6% 'other'. PMID:18070242

  19. Multi-scale biomarker evaluation of the toxicity of a commercial azo dye (Disperse Red 1) in an animal model, the freshwater cnidarian Hydra attenuata.

    PubMed

    de Jong, Laetitia; Pech, Nicolas; de Aragão Umbuzeiro, Gisela; Moreau, Xavier

    2016-06-01

    Acute (24 h, 48 h, 72 h) and chronic (7 days) tests have been performed to evaluate the effects of the commercial azo dye Disperse Red 1 (DR1) using various biomarkers in the freshwater invertebrate Hydra attenuata. Morphological changes have been selected to calculate ecotoxicological thresholds for sublethal and lethal DR1 concentrations. A multinomial logistic model showed that the probability of each morphological stage occurrence was function of concentration, time and interaction between both. Results of oxidative balance parameter measurements (72 h and 7 days) suggest that polyps set up defense mechanisms to limit lipid peroxidation caused by DR1. DR1 exposure at hormetic concentrations induces increase of asexual reproductive rates. This result suggests (1) an impact on the fitness-related phenotypical traits and (2) trade-offs between reproduction and maintenance to allow the population to survive harsher conditions. Changes in serotonin immuno-labeling in polyps showing alterations in feeding behavior suggest that chronic DR1 exposure impaired neuronal processes related to ingesting behavior in H. attenuata. This ecotoxicity study sheds light on the possible serotonin function in Hydra model and reports for the first time that serotonin could play a significant role in feeding behavior. This study used a multi-scale biomarker approach investigating biochemical, morphological, reproductive and behavioral endpoints in Hydra attenuata. This organism is proposed for a pertinent animal model to assess ecotoxicological impact of pollutant mixtures in freshwater environment. PMID:27019466

  20. Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T-DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana.

    PubMed

    Chen, Ke; Dorlhac de Borne, François; Szegedi, Ernö; Otten, Léon

    2014-11-01

    Nicotiana species carry cellular T-DNA sequences (cT-DNAs), acquired by Agrobacterium-mediated transformation. We characterized the cT-DNA sequences of the ancestral Nicotiana tabacum species Nicotiana tomentosiformis by deep sequencing. N. tomentosiformis contains four cT-DNA inserts derived from different Agrobacterium strains. Each has an incomplete inverted-repeat structure. TA is similar to part of the Agrobacterium rhizogenes 1724 mikimopine-type T-DNA, but has unusual orf14 and mis genes. TB carries a 1724 mikimopine-type orf14-mis fragment and a mannopine-agropine synthesis region (mas2-mas1-ags). The mas2' gene codes for an active enzyme. TC is similar to the left part of the A. rhizogenes A4 T-DNA, but also carries octopine synthase-like (ocl) and c-like genes normally found in A. tumefaciens. TD shows a complex rearrangement of T-DNA fragments similar to the right end of the A4 TL-DNA, and including an orf14-like gene and a gene with unknown function, orf511. The TA, TB, TC and TD insertion sites were identified by alignment with N. tabacum and Nicotiana sylvestris sequences. The divergence values for the TA, TB, TC and TD repeats provide an estimate for their relative introduction times. A large deletion has occurred in the central part of the N. tabacum cv. Basma/Xanthi TA region, and another deletion removed the complete TC region in N. tabacum. Nicotiana otophora lacks TA, TB and TD, but contains TC and another cT-DNA, TE. This analysis, together with that of Nicotiana glauca and other Nicotiana species, indicates multiple sequential insertions of cT-DNAs during the evolution of the genus Nicotiana.

  1. Large-scale detection and application of expressed sequence tag single nucleotide polymorphisms in Nicotiana.

    PubMed

    Wang, Y; Zhou, D; Wang, S; Yang, L

    2015-01-01

    Single nucleotide polymorphisms (SNPs) are widespread in the Nicotiana genome. Using an alignment and variation detection method, we developed 20,607,973 SNPs, based on the expressed sequence tag sequences of 10 Nicotiana species. The replacement rate was much higher than the transversion rate in the SNPs, and SNPs widely exist in the Nicotiana. In vitro verification indicated that all of the SNPs were high quality and accurate. Evolutionary relationships between 15 varieties were investigated by polymerase chain reaction with a special primer; the specific 302 locus of these sequence results clearly indicated the origin of Zhongyan 100. A database of Nicotiana SNPs (NSNP) was developed to store and search for SNPs in Nicotiana. NSNP is a tool for researchers to develop SNP markers of sequence data. PMID:26214460

  2. Large-scale detection and application of expressed sequence tag single nucleotide polymorphisms in Nicotiana.

    PubMed

    Wang, Y; Zhou, D; Wang, S; Yang, L

    2015-07-14

    Single nucleotide polymorphisms (SNPs) are widespread in the Nicotiana genome. Using an alignment and variation detection method, we developed 20,607,973 SNPs, based on the expressed sequence tag sequences of 10 Nicotiana species. The replacement rate was much higher than the transversion rate in the SNPs, and SNPs widely exist in the Nicotiana. In vitro verification indicated that all of the SNPs were high quality and accurate. Evolutionary relationships between 15 varieties were investigated by polymerase chain reaction with a special primer; the specific 302 locus of these sequence results clearly indicated the origin of Zhongyan 100. A database of Nicotiana SNPs (NSNP) was developed to store and search for SNPs in Nicotiana. NSNP is a tool for researchers to develop SNP markers of sequence data.

  3. Molecular characterization of quinolinate phosphoribosyltransferase (QPRtase) in Nicotiana.

    PubMed

    Sinclair, S J; Murphy, K J; Birch, C D; Hamill, J D

    2000-11-01

    Quinolate acid phosphoribosyltransferase (QPRTase), a key enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, also plays an important role in ensuring nicotinic acid is available for the synthesis of defensive pyridine alkaloids in Nicotiana species. In this study, cDNAs for QPRTase were characterized from N. rustica and N. tabacum. Deduced proteins from both cDNAs are almost identical and contain a 24 amino acid N-terminal extension, not reported in other QPRTases, that has characteristics of a mitochondrial targeting sequence. In N. tabacum and N. sylvestris, both of which contain nicotine as the major pyridine alkaloid, QPRTase transcript was detected in roots, the site of nicotine synthesis, but not in leaves. QPRTase transcript levels increased markedly in roots of both species 12-24 h after damage to aerial tissues, with a concomitant rise in transcript levels of putrescine N-methyltransferase (PMT), another key enzyme in nicotine biosynthesis. In N. glauca, however, in which anabasine represents the major pyridine alkaloid, QPRTase transcript was detected in both leaf and root tissues. Moreover, wound induction of QPRTase but not PMT was observed in leaf tissues, and not in roots, 12-24 h after wounding. Southern analysis of genomic DNA from the Nicotiana species noted above, and also several others from within the genus, suggested that QPRTase is encoded by a small gene family in all the species investigated. PMID:11198422

  4. Complete Chloroplast Genome of Nicotiana otophora and its Comparison with Related Species.

    PubMed

    Asaf, Sajjad; Khan, Abdul L; Khan, Abdur R; Waqas, Muhammad; Kang, Sang-Mo; Khan, Muhammad A; Lee, Seok-Min; Lee, In-Jung

    2016-01-01

    Nicotiana otophora is a wild parental species of Nicotiana tabacum, an interspecific hybrid of Nicotiana tomentosiformis and Nicotiana sylvestris. However, N. otophora is least understood as an alternative paternal donor. Here, we compared the fully assembled chloroplast (cp) genome of N. otophora and with those of closely related species. The analysis showed a cp genome size of 156,073 bp and exhibited a typical quadripartite structure, which contains a pair of inverted repeats separated by small and large single copies, containing 163 representative genes, with 165 microsatellites distributed unevenly throughout the genome. Comparative analysis of a gene with known function across Nicotiana species revealed 76 protein-coding sequences, 20 tRNA sequences, and 3 rRNA sequence shared between the cp genomes. The analysis revealed that N. otophora is a sister species to N. tomentosiformis within the Nicotiana genus, and Atropha belladonna and Datura stramonium are their closest relatives. These findings provide a valuable analysis of the complete N. otophora cp genome, which can identify species, elucidate taxonomy, and reconstruct the phylogeny of genus Nicotiana. PMID:27379132

  5. Complete Chloroplast Genome of Nicotiana otophora and its Comparison with Related Species

    PubMed Central

    Asaf, Sajjad; Khan, Abdul L.; Khan, Abdur R.; Waqas, Muhammad; Kang, Sang-Mo; Khan, Muhammad A.; Lee, Seok-Min; Lee, In-Jung

    2016-01-01

    Nicotiana otophora is a wild parental species of Nicotiana tabacum, an interspecific hybrid of Nicotiana tomentosiformis and Nicotiana sylvestris. However, N. otophora is least understood as an alternative paternal donor. Here, we compared the fully assembled chloroplast (cp) genome of N. otophora and with those of closely related species. The analysis showed a cp genome size of 156,073 bp and exhibited a typical quadripartite structure, which contains a pair of inverted repeats separated by small and large single copies, containing 163 representative genes, with 165 microsatellites distributed unevenly throughout the genome. Comparative analysis of a gene with known function across Nicotiana species revealed 76 protein-coding sequences, 20 tRNA sequences, and 3 rRNA sequence shared between the cp genomes. The analysis revealed that N. otophora is a sister species to N. tomentosiformis within the Nicotiana genus, and Atropha belladonna and Datura stramonium are their closest relatives. These findings provide a valuable analysis of the complete N. otophora cp genome, which can identify species, elucidate taxonomy, and reconstruct the phylogeny of genus Nicotiana. PMID:27379132

  6. Toxicity of polychlorinated diphenyl ethers in hydra attenuata and in rat whole-embryo culture. Master's thesis

    SciTech Connect

    Becker, M.C.

    1991-05-01

    Polychlorinated diphenyl ethers (PCDEs) are a class of biaryl compounds that have little commercial application, but appear to be widespread in the environment. They have been found in wood preservative waste dumpsites and in fly ash from municipal waste incinerators. They have been detected in bird eggs and tissues, fish, and other edible marine organisms in the United States, Canada, and Europe. There are limited reports in the extant literature on the toxicity of PCDEs. This study was designed to evaluate the toxicity of selected PCDEs in cultures of Hydra attenuata and post-implantation rat whole embryos. The toxicity of several closely related polychlorinated biphenyls (PCBs) was evaluated in both cultures and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was evaluated in whole embryo culture. Embryonic growth and development parameters (yolk sac diameter, crown-rump length, somite count, and DNA and protein content) and gross morphology were determined. Findings indicated that these chemicals were neither embryotoxic nor teratogenic. Thus, the PCDEs, which elicit other diverse toxic and biochemical responses in rodents, are relatively inactive in these bioassays for developmental toxicity.

  7. On the development of Cetacean extremities: II. Morphogenesis and histogenesis of the flippers in the spotted dolphin (Stenella attenuata).

    PubMed

    Sedmera, D; Misek, I; Klima, M

    1997-04-01

    Externally, the flippers of Cetacea resemble fish fins, but their internal structure is entirely mammalian. They show, however, some adaptative deviations from the typical pattern of the mammalian extremities, the most striking of which is an increased number of phalanges. The aim of this study is to describe the course of the development of flippers in the spotted dolphin (Stenella attenuata) and compare its features with other similar species from an evolutionary perspective. Early stages of flipper development were studied histologically. Differentiation of cartilaginous anlagens of the skeleton progresses proximodistally, condensation in digital rays being evident sooner than chondrogenesis in the carpal region. In one specimen, the temporary presence of cartilaginous rudiments of two carpal elements, which are not found in adults, was observed. At all examined stages, phalangeal number progressively increases up to (radial to ulnar) 3, 7, 7, 5, 3 in the most advanced stage. The reason for this condition is the specialised function of these limb-like structures. It is a classical example of convergence, in which mammalian extremities change their form to emulate the fin function. A similar condition is found in another group of originally terrestrial animals secondarily fully adapted to the aquatic mode of life-Ichyosauria (Reptilia). PMID:9253589

  8. Live CT imaging of sound reception anatomy and hearing measurements in the pygmy killer whale, Feresa attenuata.

    PubMed

    Montie, Eric W; Manire, Charlie A; Mann, David A

    2011-03-15

    In June 2008, two pygmy killer whales (Feresa attenuata) were stranded alive near Boca Grande, FL, USA, and were taken into rehabilitation. We used this opportunity to learn about the peripheral anatomy of the auditory system and hearing sensitivity of these rare toothed whales. Three-dimensional (3-D) reconstructions of head structures from X-ray computed tomography (CT) images revealed mandibles that were hollow, lacked a bony lamina medial to the pan bone and contained mandibular fat bodies that extended caudally and abutted the tympanoperiotic complex. Using auditory evoked potential (AEP) procedures, the modulation rate transfer function was determined. Maximum evoked potential responses occurred at modulation frequencies of 500 and 1000 Hz. The AEP-derived audiograms were U-shaped. The lowest hearing thresholds occurred between 20 and 60 kHz, with the best hearing sensitivity at 40 kHz. The auditory brainstem response (ABR) was composed of seven waves and resembled the ABR of the bottlenose and common dolphins. By changing electrode locations, creating 3-D reconstructions of the brain from CT images and measuring the amplitude of the ABR waves, we provided evidence that the neuroanatomical sources of ABR waves I, IV and VI were the auditory nerve, inferior colliculus and the medial geniculate body, respectively. The combination of AEP testing and CT imaging provided a new synthesis of methods for studying the auditory system of cetaceans. PMID:21346122

  9. Evolutionary potential and adaptation of Banksia attenuata (Proteaceae) to climate and fire regime in southwestern Australia, a global biodiversity hotspot

    NASA Astrophysics Data System (ADS)

    He, Tianhua; D’Agui, Haylee; Lim, Sim Lin; Enright, Neal J.; Luo, Yiqi

    2016-05-01

    Substantial climate changes are evident across Australia, with declining rainfall and rising temperature in conjunction with frequent fires. Considerable species loss and range contractions have been predicted; however, our understanding of how genetic variation may promote adaptation in response to climate change remains uncertain. Here we characterized candidate genes associated with rainfall gradients, temperatures, and fire intervals through environmental association analysis. We found that overall population adaptive genetic variation was significantly affected by shortened fire intervals, whereas declining rainfall and rising temperature did not have a detectable influence. Candidate SNPs associated with rainfall and high temperature were diverse, whereas SNPs associated with specific fire intervals were mainly fixed in one allele. Gene annotation further revealed four genes with functions in stress tolerance, the regulation of stomatal opening and closure, energy use, and morphogenesis with adaptation to climate and fire intervals. B. attenuata may tolerate further changes in rainfall and temperature through evolutionary adaptations based on their adaptive genetic variation. However, the capacity to survive future climate change may be compromised by changes in the fire regime.

  10. Evolutionary potential and adaptation of Banksia attenuata (Proteaceae) to climate and fire regime in southwestern Australia, a global biodiversity hotspot

    PubMed Central

    He, Tianhua; D’Agui, Haylee; Lim, Sim Lin; Enright, Neal J.; Luo, Yiqi

    2016-01-01

    Substantial climate changes are evident across Australia, with declining rainfall and rising temperature in conjunction with frequent fires. Considerable species loss and range contractions have been predicted; however, our understanding of how genetic variation may promote adaptation in response to climate change remains uncertain. Here we characterized candidate genes associated with rainfall gradients, temperatures, and fire intervals through environmental association analysis. We found that overall population adaptive genetic variation was significantly affected by shortened fire intervals, whereas declining rainfall and rising temperature did not have a detectable influence. Candidate SNPs associated with rainfall and high temperature were diverse, whereas SNPs associated with specific fire intervals were mainly fixed in one allele. Gene annotation further revealed four genes with functions in stress tolerance, the regulation of stomatal opening and closure, energy use, and morphogenesis with adaptation to climate and fire intervals. B. attenuata may tolerate further changes in rainfall and temperature through evolutionary adaptations based on their adaptive genetic variation. However, the capacity to survive future climate change may be compromised by changes in the fire regime. PMID:27210077

  11. Evolutionary potential and adaptation of Banksia attenuata (Proteaceae) to climate and fire regime in southwestern Australia, a global biodiversity hotspot.

    PubMed

    He, Tianhua; D'Agui, Haylee; Lim, Sim Lin; Enright, Neal J; Luo, Yiqi

    2016-05-23

    Substantial climate changes are evident across Australia, with declining rainfall and rising temperature in conjunction with frequent fires. Considerable species loss and range contractions have been predicted; however, our understanding of how genetic variation may promote adaptation in response to climate change remains uncertain. Here we characterized candidate genes associated with rainfall gradients, temperatures, and fire intervals through environmental association analysis. We found that overall population adaptive genetic variation was significantly affected by shortened fire intervals, whereas declining rainfall and rising temperature did not have a detectable influence. Candidate SNPs associated with rainfall and high temperature were diverse, whereas SNPs associated with specific fire intervals were mainly fixed in one allele. Gene annotation further revealed four genes with functions in stress tolerance, the regulation of stomatal opening and closure, energy use, and morphogenesis with adaptation to climate and fire intervals. B. attenuata may tolerate further changes in rainfall and temperature through evolutionary adaptations based on their adaptive genetic variation. However, the capacity to survive future climate change may be compromised by changes in the fire regime.

  12. Cadmium contamination of tissues and organs of delphinids species (Stenella attenuata)--influence of biological and ecological factors

    SciTech Connect

    Andre, J.M.; Amiard, J.C.; Amiard-Triquet, C.; Boudou, A.; Ribeyre, F. )

    1990-12-01

    Based on a sample of 27 dolphins (Stenella attenuata) captured in the Eastern tropical zone of the Pacific Ocean, this study was carried out to analyze the cadmium accumulation levels and distribution in 12 organs or tissue samples. The average cadmium concentrations were between 0.2 mg Cd.kg-1 in the brain and muscle and 48 mg Cd.kg-1 in the kidneys. For most of organs and tissues the average values were between 1 and 5 mg Cd.kg-1. Kidneys, liver, muscle, and intestine contained almost 85% of the total cadmium burden of all tissues considered in this study. Most of the biological and ecological factors taken into account (age, sex, total weight, and length of the dolphins, weight of the organs, place and date of capture) interacted with the cadmium concentrations and burdens in the collected organs or tissues. Three factors appear to be of prime importance: age, body weight, and geographical location of the area of capture.

  13. PHANTASTICA regulates development of the adaxial mesophyll in Nicotiana leaves.

    PubMed

    McHale, Neil A; Koning, Ross E

    2004-05-01

    Initiation and growth of leaf blades is oriented by an adaxial/abaxial axis aligned with the original axis of polarity in the leaf primordium. To investigate mechanisms regulating this process, we cloned the Nicotiana tabacum ortholog of PHANTASTICA (NTPHAN) and generated a series of antisense transgenics in N. sylvestris. We show that NSPHAN is expressed throughout emerging blade primordia in the wild type and becomes localized to the middle mesophyll in the expanding lamina. Antisense NSPHAN leaves show ectopic expression of NTH20, a class I KNOX gene. Juvenile transgenic leaves have normal adaxial/abaxial polarity and generate leaf blades in the normal position, but the adaxial mesophyll shows disorganized patterns of cell division, delayed maturation of palisade, and ectopic reinitiation of blade primordia along the midrib. Reversal of the phenotype with exogenous gibberellic acid suggests that NSPHAN, acting via KNOX repression, maintains determinacy in the expanding lamina and sustains the patterns of cell proliferation critical to palisade development.

  14. Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

    PubMed Central

    Barankiewicz, J; Paszkowski, J

    1980-01-01

    The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed. PMID:6154458

  15. Cell Death Processes during Expression of Hybrid Lethality in Interspecific F1 Hybrid between Nicotiana gossei Domin and Nicotiana tabacum

    PubMed Central

    Mino, Masanobu; Maekawa, Kenji; Ogawa, Ken'ichi; Yamagishi, Hiroshi; Inoue, Masayoshi

    2002-01-01

    Hybrid lethality, a type of reproductive isolation, is a genetically controlled event appearing at the seedling stage in interspecific hybrids. We characterized the lethality of F1 hybrid seedlings from Nicotiana gossei Domin and Nicotiana tabacum cv Bright-Yellow 4 using a number of traits including growth rate, microscopic features of tissues and cells, ion leakage, DNA degradation, reactive oxygen intermediates including superoxide radical (O2−) and hydrogen peroxide (H2O2), and expression of stress response marker genes. Lethal symptoms appeared at 4 d after germination in the basal hypocotyl and extended toward both the hypocotyl and root of the plants grown at 26°C. Microscopic analysis revealed a prompt lysis of cell components during cell death. Membrane disruption and DNA degradation were found in the advanced stage of the lethality. The death of mesophyll cells in the cotyledon was initiated by the vascular bundle, suggesting that a putative factor inducing cell death diffused into surrounding cells from the vascular tissue. In contrast, these symptoms were not observed in the plants grown at 37°C. Seedlings grown at 26°C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37°C. A number of stress response marker genes were expressed at 26°C but not at 37°C. We proposed that a putative death factor moving systemically through the vascular system induced a prompt and successive lysis of the cytoplasm of cells and that massive cell death eventually led to the loss of the hybrid plant. PMID:12481061

  16. [Occurrence and behavioral patterns of the spotted coastal dolphin Stenella attenuata (Cetacea: delphinidae) in the Gulf of Papagayo, Costa Rica].

    PubMed

    May-Collado, Laura; Ramírez, Alvaro Morales

    2005-01-01

    Dolphins are characterized by a significant behavioral versatility, which allows them to respond to environmental seasonality. Seasonal variation in dolphin behavior in tropical waters is not well known. Stenella attenuata graffmani is a resident dolphin in the clearly defined seasonal Gulf of Papagayo, Costa Rica, and we studied if dolphin group size, occurrence and behavioral patterns were associated with season and time of day in the gulf. Using strip transects we surveyed two locations for three consecutive years. School size ranged from 1 to 50 individuals, mean group size was 10.16 (SD = 9.61) individuals. Overall, foraging activities were the most frequent, followed by social interactions and travel. From 6:00 AM to 9:00 AM we mostly observed social interactions, followed by feeding-socializing (9:00 AM-12:00 PM) and feeding exclusively (12:00 PM-3:00 PM). Social activities intensified afterwards (3:00 PM-6:00 PM). Behavior and gulf seasonality were associated (chi2 = 90.52, gl = 6, p<0.05, n = 99). In the dry season (December-April) feeding predominated over other activities, but socializing was more frequent in the early rainy season (May-July). Larger groups (mean 12 dolphins) forage actively; smaller groups (mean 6 dolphins 6.51 +/- 5.12) foraged more passively. Seasonal variation in dolphin activities are likely to be associated with food availability, as observed in the high number of groups involved in foraging behaviors, and a high investment in foraging activities during the dry season. PMID:17354439

  17. Glyphosate Tolerance in Tobacco (Nicotiana tabacum L.) 1

    PubMed Central

    Dyer, William E.; Weller, Stephen C.; Bressan, Ray A.; Herrmann, Klaus M.

    1988-01-01

    A glyphosate-tolerant tobacco cell line, Nicotiana tabacum L. Indiana (I7), was selected from the glyphosate-sensitive Wisconsin 38 (W38) line through a single step exposure to the herbicide. Tolerance and growth characteristics of I7 cells were the same for cells maintained for more than 1 year in the presence or absence of glyphosate. Glyphosate tolerance levels were constant through the growth cycle. Tolerance is not due to reduced uptake of glyphosate. Shikimate levels in I7 and W38 cells maintained in glyphosate-free medium were similar, whereas W38 cells accumulated 46 times more shikimate than I7 cells, when cells of both lines were exposed to the herbicide. Glyphosate treatment caused increased levels of aromatic amino acids in W38 cells and slightly lower levels in I7 cells. Specific activities of dehydroquinate synthase, shikimate dehydrogenase, and shikimate kinase were similar in the two cell types, whereas DAHP synthase and EPSP synthase specific activities were elevated in I7 cells. Plants regenerated from I7 cells retained tolerance to glyphosate. Images Fig. 7 PMID:16666365

  18. Piriformospora indica confers cadmium tolerance in Nicotiana tabacum.

    PubMed

    Hui, Feiqiong; Liu, Jian; Gao, Qikang; Lou, Binggan

    2015-11-01

    Piriformospora indica, a root-colonizing endophytic fungus of Sebacinales, promotes plant growth and confers resistance against biotic and abiotic stresses. In order to confirm the influence of P. indica on growth, proline, malondialdehyde (MDA), chlorophyll, and cadmium (Cd) amounts in Nicotiana tabacum under Cd stress, hydroponics, pot and field trials were conducted. The results showed that P. indica can store Cd in plant roots and reduce leaf Cd content, reduce the concentration of MDA, and increase the proline and chlorophyll content and the activities of catalase, peroxidase, and superoxide dismutase under hydroponic Cd stress. RT-PCR analysis showed that the relative expression level of genes Gsh2, TaPCS1, oas1, GPX, and Hsp70 in colonized plants was 4.3, 1.4, 2.9, 1.7, and 6.9 fold higher than in un-colonized plants respectively. Cd exposure significantly reduced un-colonized plants' agronomic traits compared to P. indica-colonized ones. Our results suggested that P. indica can sequester Cd in roots, so that much less cadmium was transported to leaves, and the increased concentrations of antioxidant enzymes, pigments and proline contents, as well as the higher expression of stress-related phytochelatin biosynthesis genes in P. indica-inoculated plants, may also serve to protect N. tabacum plants against oxidative damage, enhancing Cd tolerance. PMID:26574103

  19. Efficacy of Chaetomium Species as Biological Control Agents against Phytophthora nicotianae Root Rot in Citrus

    PubMed Central

    Wattanachai, Pongnak; Kasem, Soytong; Poeaim, Supattra

    2015-01-01

    Thailand is one of the largest citrus producers in Southeast Asia. Pathogenic infection by Phytophthora, however, has become one of major impediments to production. This study identified a pathogenic oomycete isolated from rotted roots of pomelo (Citrus maxima) in Thailand as Phytophthora nicotianae by the internal transcribed spacer ribosomal DNA sequence analysis. Then, we examined the in vitro and in vivo effects of Chaetomium globosum, Chaetomium lucknowense, Chaetomium cupreum and their crude extracts as biological control agents in controlling this P. nicotianae strain. Represent as antagonists in biculture test, the tested Chaetomium species inhibited mycelial growth by 50~56% and parasitized the hyphae, resulting in degradation of P. nicotianae mycelia after 30 days. The crude extracts of these Chaetomium species exhibited antifungal activities against mycelial growth of P. nicotianae, with effective doses of 2.6~101.4 µg/mL. Under greenhouse conditions, application of spores and methanol extracts of these Chaetomium species to pomelo seedlings inoculated with P. nicotianae reduced root rot by 66~71% and increased plant weight by 72~85% compared to that in the control. The method of application of antagonistic spores to control the disease was simple and economical, and it may thus be applicable for large-scale, highly effective biological control of this pathogen. PMID:26539045

  20. A distinct endogenous pararetrovirus family in Nicotiana tomentosiformis, a diploid progenitor of polyploid tobacco.

    PubMed

    Gregor, Wolfgang; Mette, M Florian; Staginnus, Christina; Matzke, Marjori A; Matzke, Antonius J M

    2004-03-01

    A distinct endogenous pararetrovirus (EPRV) family corresponding to a previously unknown virus has been identified in the genome of Nicotiana tomentosiformis, a diploid ancestor of allotetraploid tobacco (Nicotiana tabacum). The putative virus giving rise to N. tomentosiformis EPRVs (NtoEPRVs) is most similar to tobacco vein clearing virus, an episomal form of a normally silent EPRV family in Nicotiana glutinosa; it is also related to a putative virus giving rise to the NsEPRV family in Nicotiana sylvestris (the second diploid progenitor of tobacco) and in the N. sylvestris fraction of the tobacco genome. The copy number of NtoEPRVs is significantly higher in N. tomentosiformis than in tobacco. This suggests that after the polyploidization event, many copies were lost from the polyploid genome or were accumulated specifically in the diploid genome. By contrast, the copy number of NsEPRVs has remained constant in N. sylvestris and tobacco, indicating that changes have occurred preferentially in the NtoEPRV family during evolution of the three Nicotiana species. NtoEPRVs are often flanked by Gypsy retrotransposon-containing plant DNA. Although the mechanisms of NtoEPRV integration, accumulation, and/or elimination are unknown, these processes are possibly linked to retrotransposon activity.

  1. Current status and prospects for the study of Nicotiana genomics, genetics, and nicotine biosynthesis genes.

    PubMed

    Wang, Xuewen; Bennetzen, Jeffrey L

    2015-02-01

    Nicotiana, a member of the Solanaceae family, is one of the most important research model plants, and of high agricultural and economic value worldwide. To better understand the substantial and rapid research progress with Nicotiana in recent years, its genomics, genetics, and nicotine gene studies are summarized, with useful web links. Several important genetic maps, including a high-density map of N. tabacum consisting of ~2,000 markers published in 2012, provide tools for genetics research. Four whole genome sequences are from allotetraploid species, including N. benthamiana in 2012, and three N. tabacum cultivars (TN90, K326, and BX) in 2014. Three whole genome sequences are from diploids, including progenitors N. sylvestris and N. tomentosiformis in 2013 and N. otophora in 2014. These and additional studies provide numerous insights into genome evolution after polyploidization, including changes in gene composition and transcriptome expression in N. tabacum. The major genes involved in the nicotine biosynthetic pathway have been identified and the genetic basis of the differences in nicotine levels among Nicotiana species has been revealed. In addition, other progress on chloroplast, mitochondrial, and NCBI-registered projects on Nicotiana are discussed. The challenges and prospects for genomic, genetic and application research are addressed. Hence, this review provides important resources and guidance for current and future research and application in Nicotiana.

  2. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

    PubMed

    Gao, Junping; Wang, Genhong; Ma, Sanyuan; Xie, Xiaodong; Wu, Xiangwei; Zhang, Xingtan; Wu, Yuqian; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

  3. Paenibacillus nicotianae sp. nov., isolated from a tobacco sample.

    PubMed

    Li, Qing-Qing; Zhou, Xing-Kui; Dang, Li-Zhi; Cheng, Juan; Hozzein, Wael N; Liu, Min-Jiao; Hu, Qun; Li, Wen-Jun; Duan, Yan-Qing

    2014-12-01

    A Gram-stain positive, facultative anaerobic endospore-forming bacterium, designated strain YIM h-19(T), was isolated from a tobacco sample. Cells were observed to be motile rods by means of peritrichous flagella and colonies were observed to be convex, yellow, circular and showed catalase-positive and oxidase-negative reactions. Strain YIM h-19(T) was able to grow at 4-45 °C, pH 6.0-8.0 and 0-3 % NaCl (w/v). The predominant respiratory quinone was identified as MK-7. Major fatty acids were identified as anteiso-C15:0, anteiso-C17:0 and C16:0. The polar lipids were found to be phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unidentified polar lipids. The genomic DNA G+C content was determined to be 54 mol%. 16S rRNA gene sequence analysis showed the strain YIM h-19(T) was most closely related to Paenibacillus hordei RH-N24(T) and Paenibacillus hunanensis FeL05(T) with similarities of 98.30 and 94.64 % respectively. However, DNA-DNA hybridization data indicated that the isolate represented a novel genomic species with the genus Paenibacillus. All data from genotypic and phenotypic analyses support the conclusion that strain YIM h-19(T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillus nicotianae sp. nov. is proposed. The type strain is YIM h-19(T) (=CGMCC1.12819(T) = NRRL B-59112(T)). PMID:25239270

  4. Nucleotide sequence of the tobacco (Nicotiana tabacum) anionic peroxidase gene

    SciTech Connect

    Diaz-De-Leon, F.; Klotz, K.L.; Lagrimini, L.M. )

    1993-03-01

    Peroxidases have been implicated in numerous physiological processes including lignification (Grisebach, 1981), wound-healing (Espelie et al., 1986), phenol oxidation (Lagrimini, 1991), pathogen defense (Ye et al., 1990), and the regulation of cell elongation through the formation of interchain covalent bonds between various cell wall polymers (Fry, 1986; Goldberg et al., 1986; Bradley et al., 1992). However, a complete description of peroxidase action in vivo is not available because of the vast number of potential substrates and the existence of multiple isoenzymes. The tobacco anionic peroxidase is one of the better-characterized isoenzymes. This enzyme has been shown to oxidize a number of significant plant secondary compounds in vitro including cinnamyl alcohols, phenolic acids, and indole-3-acetic acid (Maeder, 1980; Lagrimini, 1991). A cDNA encoding the enzyme has been obtained, and this enzyme was shown to be expressed at the highest levels in lignifying tissues (xylem and tracheary elements) and also in epidermal tissue (Lagrimini et al., 1987). It was shown at this time that there were four distinct copies of the anionic peroxidase gene in tobacco (Nicotiana tabacum). A tobacco genomic DNA library was constructed in the [lambda]-phase EMBL3, from which two unique peroxidase genes were sequenced. One of these clones, [lambda]POD1, was designated as a pseudogene when the exonic sequences were found to differ from the cDNA sequences by 1%, and several frame shifts in the coding sequences indicated a dysfunctional gene (the authors' unpublished results). The other clone, [lambda]POD3, described in this manuscript, was designated as the functional tobacco anionic peroxidase gene because of 100% homology with the cDNA. Significant structural elements include an AS-2 box indicated in shoot-specific expression (Lam and Chua, 1989), a TATA box, and two intervening sequences. 10 refs., 1 tab.

  5. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

    PubMed

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L; Arzola, Lucas; Lebrilla, Carlito B; Dandekar, Abhaya M; Falk, Bryce W; Nandi, Somen; Rodriguez, Raymond L; McDonald, Karen A

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  6. Nicotiana benthamiana as a Production Platform for Artemisinin Precursors

    PubMed Central

    van Herpen, Teun W. J. M.; Cankar, Katarina; Nogueira, Marilise; Bosch, Dirk; Bouwmeester, Harro J.; Beekwilder, Jules

    2010-01-01

    Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg.kg−1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed. PMID:21151979

  7. Antimicrobial Activity of Bacteriophage Endolysin Produced in Nicotiana benthamiana Plants.

    PubMed

    Kovalskaya, Natalia; Foster-Frey, Juli; Donovan, David M; Bauchan, Gary; Hammond, Rosemarie W

    2016-01-01

    The increasing spread of antibiotic-resistant pathogens has raised the interest in alternative antimicrobial treatments. In our study, the functionally active gram-negative bacterium bacteriophage CP933 endolysin was produced in Nicotiana benthamiana plants by a combination of transient expression and vacuole targeting strategies, and its antimicrobial activity was investigated. Expression of the cp933 gene in E. coli led to growth inhibition and lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of the cp933 gene in plants using Potato virus X-based transient expression vectors (pP2C2S and pGR107) resulted in death of the apical portion of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its Nterminus to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933 to the delta-type vacuoles. Plants producing the CP933 fusion protein did not exhibit the severe toxic effects seen with the unfused protein and the level of expression was 0.16 mg/g of plant tissue. Antimicrobial assays revealed that, in contrast to gram-negative bacterium E. coli (BL21(DE3)), the gram-positive plant pathogenic bacterium Clavibacter michiganensis was more susceptible to the plant-produced CP933, showing 18% growth inhibition. The results of our experiments demonstrate that the combination of transient expression and protein targeting to the delta vacuoles is a promising approach to produce functionally active proteins that exhibit toxicity when expressed in plant cells. PMID:26403819

  8. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  9. Pollination triggers female gametophyte development in immature Nicotiana tabacum flowers

    PubMed Central

    Brito, Michael S.; Bertolino, Lígia T.; Cossalter, Viviane; Quiapim, Andréa C.; DePaoli, Henrique C.; Goldman, Gustavo H.; Teixeira, Simone P.; Goldman, Maria H. S.

    2015-01-01

    In Nicotiana tabacum, female gametophytes are not fully developed at anthesis, but flower buds pollinated 12 h before anthesis produce mature embryo sacs. We investigated several pollination-associated parameters in N. tabacum flower buds to determine the developmental timing of important events in preparation for successful fertilization. First, we performed hand pollinations in flowers from stages 4 to 11 to study at which developmental stage pollination would produce fruits. A Peroxtesmo test was performed to correlate peroxidase activity on the stigma surface, indicative of stigma receptivity, with fruit set. Pollen tube growth and female gametophyte development were microscopically analyzed in pistils of different developmental stages. Fruits were obtained only after pollinations of flower buds at late stage 7 and older; fruit weight and seed germination capacity increased as the developmental stage of the pollinated flower approached anthesis. Despite positive peroxidase activity and pollen tube growth, pistils at stages 5 and 6 were unable to produce fruits. At late stage 7, female gametophytes were undergoing first mitotic division. After 24 h, female gametophytes of unpollinated pistils were still in the end of the first division, whereas those of pollinated pistils showed egg cells. RT-qPCR assay showed that the expression of the NtEC1 gene, a marker of egg cell development, is considerably higher in pollinated late stage 7 ovaries compared with unpollinated ovaries. To test whether ethylene is the signal eliciting female gametophyte maturation, the expression of ACC synthase was examined in unpollinated and pollinated stage 6 and late stage 7 stigmas/styles. Pollination induced NtACS expression in stage 6 pistils, which are unable to produce fruits. Our results show that pollination is a stimulus capable of triggering female gametophyte development in immature tobacco flowers and suggests the existence of a yet undefined signal sensed by the pistil. PMID

  10. Production, partial purification and characterization of xylanase using Nicotiana tabacum leaf dust as substrate.

    PubMed

    Acharya, Komal P; Shilpkar, Prateek

    2016-03-01

    Isolated Bacillus sp. was used in the present study for production of xylanase from Nicotiana tabacum leaf dust. The strain was able to give a maximum of 1.77 Uml⁻¹ xylanase activity under optimized fermentation conditions which was further increased upto 2.77 Uml⁻¹ after extraction and partial purification of enzyme. After partial purification, the enzyme was characterized and it gave the highest xylanase activity at pH 7.0, when 0.2 ml enzyme was incubated with 2.0% substrate (Nicotiana tabacum leaf dust) for 60 min at 60°C. Saccharification study of Nicotiana tabacum leaf dust with partially purified enzyme revealed that 18.4% reducing sugar was released in 20 hrs incubation, and TLC and HPTLC analysis showed that xylose and glucose sugars were obtained after hydrolysis of substrate. FTIR analysis confirmed decomposition of substrate. PMID:27097451

  11. Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

    PubMed Central

    Ehrenshaft, M; Daub, M E

    1994-01-01

    We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures. Images PMID:8085820

  12. Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

    PubMed

    Ehrenshaft, M; Daub, M E

    1994-08-01

    We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures.

  13. Nicotiana tabacum as model for ozone - plant surface reactions

    NASA Astrophysics Data System (ADS)

    Jud, Werner; Fischer, Lukas; Wohlfahrt, Georg; Tissier, Alain; Canaval, Eva; Hansel, Armin

    2015-04-01

    Elevated tropospheric ozone concentrations are considered a toxic threat to plants, responsible for global crop losses with associated economic costs of several billion dollars per year. The ensuing injuries have been related to the uptake of ozone through the stomatal pores and oxidative effects damaging the internal leaf tissue. A striking question of current research is the environment and plant specific partitioning of ozone loss between gas phase, stomatal or plant surface sink terms. Here we show results from ozone fumigation experiments using various Nicotiana Tabacum varieties, whose surfaces are covered with different amounts of unsaturated diterpenoids exuded by their glandular trichomes. Exposure to elevated ozone levels (50 to 150 ppbv) for 5 to 15 hours in an exceptionally clean cuvette system did neither result in a reduction of photosynthesis nor caused any visible leaf damage. Both these ozone induced stress effects have been observed previously in ozone fumigation experiments with the ozone sensitive tobacco line Bel-W3. In our case ozone fumigation was accompanied by a continuous release of oxygenated volatile organic compounds, which could be clearly associated to their condensed phase precursors for the first time. Gas phase reactions of ozone were avoided by choosing a high enough gas exchange rate of the plant cuvette system. In the case of the Ambalema variety, that is known to exude only the diterpenoid cis-abienol, ozone fumigation experiments yield the volatiles formaldehyde and methyl vinyl ketone (MVK). The latter could be unequivocally separated from isomeric methacrolein (MACR) by the aid of a Selective Reagent Ion Time-of-Flight Mass Spectrometer (SRI-ToF-MS), which was switched every six minutes from H3O+ to NO+ primary ion mode and vice versa. Consistent with the picture of an ozone protection mechanism caused by reactive diterpenoids at the leaf surface are the results from dark-light experiments. The ozone loss obtained from the

  14. Assessing acute toxicities of pre- and post-treatment industrial wastewaters with Hydra attenuata: A comparative study of acute toxicity with the fathead minnow, Pimephales promelas

    SciTech Connect

    Fu, L.J.; Staples, R.E.; Stahl, R.G. Jr. . Haskell Lab. for Toxicology and Industrial Medicine)

    1994-04-01

    This study was undertaken to (a) determine wastewater treatment effectiveness using two freshwater organisms, (b) compare acute toxicity results from the two species exposed to the wastewaters, and (c) link acute and potential developmental toxicity of wastewaters in one organism. The acute toxicities of several pretreatment and post-treatment industrial waste-water samples wee evaluated with adult Hydra attenuata and fathead minnows. The acute LC50s agreed closely when results in Hydra attenuata were compared with those from fathead minnow tests. Acute LC50s ranged from 3 to >100% of samples with hydra, and from 1.0 to >100% of sample with fathead minnows. The results provided strong evidence of treatment effectiveness because toxicity decreased with progressive stages of treatment. Previously the Hydra Developmental Toxicity Assay was used as a prescreen mainly for in vitro assessment of developmental toxicity with pure compounds and to prioritized toxicants according to selective toxicity to the developing embryo. Recently the authors modified the assay for testing natural waters and wastewaters; hence, some of the wastewater samples also were tested for their developmental toxicity. In this case, the relative selective toxicity of these wastewater samples ranged from 0.7 to 2.1, indicating that no sample was uniquely toxic to the developing embryo, although acute toxicity was manifested. Overall, their results indicate the Hydra Assay functions appropriately in assessments of acute and developmental toxicity of industrial wastewaters and may be a simple and useful tool in a battery of tests for broader scale detection of environmental hazards.

  15. Genetic characterization of Phytophthora nicotianae by the analysis of polymorphic regions of the mitochondrial DNA.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new method based on the analysis of mitochondrial intergenic regions characterized by intraspecific variation in DNA sequences was developed and applied to the study of the plant pathogen Phytophthora nicotianae. Two regions flanked by genes trny and rns and trnw and cox2 were identified by compa...

  16. Genetic Analysis of Phytophthora nicotianae Populations from Different Hosts Using Microsatellite Markers.

    PubMed

    Biasi, Antonio; Martin, Frank N; Cacciola, Santa O; di San Lio, Gaetano Magnano; Grünwald, Niklaus J; Schena, Leonardo

    2016-09-01

    In all, 231 isolates of Phytophthora nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa, and Ruta), and other plant genera were characterized using simple-sequence repeat markers. In total, 99 multilocus genotypes (MLG) were identified, revealing a strong association between genetic grouping and host of recovery, with most MLG being associated with a single host genus. Significant differences in the structure of populations were revealed but clonality prevailed in all populations. Isolates from Citrus were found to be genetically related regardless of their geographic origin and were characterized by high genetic uniformity and high inbreeding coefficients. Higher variability was observed for other populations and a significant geographical structuring was determined for isolates from Nicotiana. Detected differences were related to the propagation and cultivation systems of different crops. Isolates obtained from Citrus spp. are more likely to be distributed worldwide with infected plant material whereas Nicotiana and Lycopersicon spp. are propagated by seed, which would not contribute to the spread of the pathogen and result in a greater chance for geographic isolation of lineages. With regard to ornamental species in nurseries, the high genetic variation is likely the result of the admixture of diverse pathogen genotypes through the trade of infected plant material from various geographic origins, the presence of several hosts in the same nursery, and genetic recombination through sexual reproduction of this heterothallic species. PMID:27111805

  17. Relationship between Active Oxygen Species, Lipid Peroxidation, Necrosis, and Phytoalexin Production Induced by Elicitins in Nicotiana.

    PubMed Central

    Rusterucci, C.; Stallaert, V.; Milat, M. L.; Pugin, A.; Ricci, P.; Blein, J. P.

    1996-01-01

    Excised leaves of Nicotiana tabacum var Xanthi and Nicotiana rustica were treated with cryptogein and capsicein, basic and acidic elicitins, respectively. Both compounds induced leaf necrosis, the intensity of which depended on concentration and duration of treatment. N. tabacum var Xanthi was the most sensitive species and cryptogein was the most active elicitin. Lipid peroxidation in elicitin-treated Nicotiana leaves was closely correlated with the appearance of necrosis. Elicitin treatments induced a rapid and transient burst of active oxygen species (AOS) in cell cultures of both Nicotiana species, with the production by Xanthi cells being 6-fold greater than that by N. rustica. Similar maximum AOS production levels were observed with both elicitins, but capsicein required 10-fold higher concentrations than those of cryptogein. Phytoalexin production was lower in response to both elicitins in N. tabacum var Xanthi cells than in N. rustica cells, and capsicein was the most efficient elicitor of this response. In cryptogein-treated cell suspensions, phytoalexin synthesis was unaffected by diphenyleneiodonium, which inhibited AOS generation, nor was it affected by tiron or catalase, which suppressed AOS accumulation in the extracellular medium. These results suggest that AOS production, lipid peroxidation, and necrosis are directly related, whereas phytoalexin production depends on neither the presence nor the intensity of these responses. PMID:12226334

  18. Physiological and molecular changes during opening and senescence of Nicotiana mutabilis flowers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The flowers of Nicotiana mutabilis, a tobacco species recently discovered in southern Brazil, have petals that undergo a striking colour change from white through pink to red as they open and senesce over a typical 7-d lifespan. Colouration in petals was associated with an increase in chalcone synt...

  19. PHYSIOLOGICAL AND MOLECULAR CHANGES DURING OPENING AND SENESCENCE OF NICOTIANA MUTABILIS FLOWERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The flowers of Nicotiana mutabilis, a tobacco species recently discovered in southern Brazil, have petals that undergo a striking colour change from white through pink to red as they open and senesce over a typical 7-d lifespan. Colouration in petals was associated with an increase in chalcone synt...

  20. Effects of hydrostatic pressure, agitation and CO2 stress on Phytophthora nicotianae zoospore survival

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytophthora nicotianae Breda de Haan was used as a model pathogen to investigate the effects of hydrostatic pressure, agitation, and aeration with CO2 or breathable air on the survival of Phytophthora zoospores in water. Injecting CO2 into 2 liters of zoospore-infested water for 5 min at 110.4 ml ...

  1. Infection of Citrus Roots by Tylenchulus semipenetrans Reduces Root Infection by Phytophthora nicotianae

    PubMed Central

    El-Borai, F. E.; Duncan, L. W.; Graham, J. H.

    2002-01-01

    Bioassays and whole-plant experiments were conducted to investigate the interaction between Tylenchulus semipenetrans and Phytophthora nicotianae. Both organisms are parasites of the citrus fibrous root cortex. Nematode-infected and non-infected root segments were excised from naturally infected field roots and placed on water agar in close proximity to agar plugs of P. nicotianae and then transferred to a Phytophthora-selective medium. At 10 and 12 days, 50% fewer nematode-infected segments were infected by P. nicotianae than non-infected segments. In whole-plant experiments in glass test tubes, sour orange seedlings were inoculated with two densities (8,000 or 80,000 eggs and second-stage juveniles) of T. semipenetrans, and after establishment of infection were inoculated with two densities (9,000 and 90,000 zoospores) of P. nicotianae. In the first experiment, fungal protein was 53% to 65% lower in the roots infected by both organisms than in roots infected by the fungus only. Compared to plants infected only by P. nicotianae, shoot weights were 33% to 50% greater (P ≤ 0.05) in plants infected by both parasites, regardless of inoculum density. Fibrous and tap root weights were 5% to 23% and 19% to 34% greater (P ≤ 0.05), respectively, in nematode-fungus combination treatments compared to the fungus alone. A second experiment was conducted, where plants were infected by the fungus, the nematode, both organisms, or neither organism. The soil mixture pH for 50% of the plants was adjusted from 4.5 to 7.0 to favor nematode infection. A higher rate of nematode infection of plants growing at pH 7.0 compared to pH 4.5 resulted in greater suppression of fungal development and greater inhibition of fungal damage to the plant. Compared to plants infected only by P. nicotianae, shoot and root weights were 37% and 33% greater (P ≤ 0.05), respectively, in plants infected by both parasites. These experiments have revealed antagonism between T. semipenetrans and P

  2. Screening micro-organisms for cadmium absorption from aqueous solution and cadmium absorption properties of Arthrobacter nicotianae.

    PubMed

    Tsuruta, Takehiko; Umenai, Daishi; Hatano, Tomonobu; Hirajima, Tsuyoshi; Sasaki, Keiko

    2014-01-01

    To obtain basic information on how microbial cells absorb cadmium from aqueous solution, we examined cadmium absorption in various micro-organisms. Of 51 micro-organism strains tested, we found that some Gram-positive bacteria, such as, Arthrobacter nicotianae and Bacillus subtilis, and some actinomycetes, such as, Streptomyces flavoviridis and S. levoris were highly capable of absorbing cadmium from an aqueous solution. A. nicotianae absorbed the largest amount of cadmium, over 800 μmol cadmium per gram of dry wt. cells. However, cadmium absorption by A. nicotianae was affected by the solution pH, cadmium concentration, and cell density. The absorption of cadmium was very rapid. Some factors that affected cadmium absorption by A. nicotianae cells were also discussed.

  3. BABA and Phytophthora nicotianae Induce Resistance to Phytophthora capsici in Chile Pepper (Capsicum annuum)

    PubMed Central

    Stamler, Rio A.; Holguin, Omar; Dungan, Barry; Schaub, Tanner; Sanogo, Soumaila; Goldberg, Natalie; Randall, Jennifer J.

    2015-01-01

    Induced resistance in plants is a systemic response to certain microorganisms or chemicals that enhances basal defense responses during subsequent plant infection by pathogens. Inoculation of chile pepper with zoospores of non-host Phytophthora nicotianae or the chemical elicitor beta-aminobutyric acid (BABA) significantly inhibited foliar blight caused by Phytophthora capsici. Tissue extract analyses by GC/MS identified conserved change in certain metabolite concentrations following P. nicotianae or BABA treatment. Induced chile pepper plants had reduced concentrations of sucrose and TCA cycle intermediates and increased concentrations of specific hexose-phosphates, hexose-disaccharides and amino acids. Galactose, which increased significantly in induced chile pepper plants, was shown to inhibit growth of P. capsici in a plate assay. PMID:26020237

  4. Nicotiana Roots Recruit Rare Rhizosphere Taxa as Major Root-Inhabiting Microbes.

    PubMed

    Saleem, Muhammad; Law, Audrey D; Moe, Luke A

    2016-02-01

    Root-associated microbes have a profound impact on plant health, yet little is known about the distribution of root-associated microbes among different root morphologies or between rhizosphere and root environments. We explore these issues here with two commercial varieties of burley tobacco (Nicotiana tabacum) using 16S rRNA gene amplicon sequencing from rhizosphere soil, as well as from primary, secondary, and fine roots. While rhizosphere soils exhibited a fairly rich and even distribution, root samples were dominated by Proteobacteria. A comparison of abundant operational taxonomic units (OTUs) between rhizosphere and root samples indicated that Nicotiana roots select for rare taxa (predominantly Proteobacteria, Verrucomicrobia, Actinobacteria, Bacteroidetes, and Acidobacteria) from their corresponding rhizosphere environments. The majority of root-inhabiting OTUs (~80 %) exhibited habitat generalism across the different root morphological habitats, although habitat specialists were noted. These results suggest a specific process whereby roots select rare taxa from a larger community.

  5. BABA and Phytophthora nicotianae Induce Resistance to Phytophthora capsici in Chile Pepper (Capsicum annuum).

    PubMed

    Stamler, Rio A; Holguin, Omar; Dungan, Barry; Schaub, Tanner; Sanogo, Soumaila; Goldberg, Natalie; Randall, Jennifer J

    2015-01-01

    Induced resistance in plants is a systemic response to certain microorganisms or chemicals that enhances basal defense responses during subsequent plant infection by pathogens. Inoculation of chile pepper with zoospores of non-host Phytophthora nicotianae or the chemical elicitor beta-aminobutyric acid (BABA) significantly inhibited foliar blight caused by Phytophthora capsici. Tissue extract analyses by GC/MS identified conserved change in certain metabolite concentrations following P. nicotianae or BABA treatment. Induced chile pepper plants had reduced concentrations of sucrose and TCA cycle intermediates and increased concentrations of specific hexose-phosphates, hexose-disaccharides and amino acids. Galactose, which increased significantly in induced chile pepper plants, was shown to inhibit growth of P. capsici in a plate assay.

  6. Transgressive phenotypes and generalist pollination in the floral evolution of Nicotiana polyploids.

    PubMed

    McCarthy, Elizabeth W; Chase, Mark W; Knapp, Sandra; Litt, Amy; Leitch, Andrew R; Le Comber, Steven C

    2016-08-08

    Polyploidy is an important driving force in angiosperm evolution, and much research has focused on genetic, epigenetic and transcriptomic responses to allopolyploidy. Nicotiana is an excellent system in which to study allopolyploidy because half of the species are allotetraploids of different ages, allowing us to examine the trajectory of floral evolution over time. Here, we study the effects of allopolyploidy on floral morphology in Nicotiana, using corolla tube measurements and geometric morphometrics to quantify petal shape. We show that polyploid morphological divergence from the intermediate phenotype expected (based on progenitor morphology) increases with time for floral limb shape and tube length, and that most polyploids are distinct or transgressive in at least one trait. In addition, we show that polyploids tend to evolve shorter and wider corolla tubes, suggesting that allopolyploidy could provide an escape from specialist pollination via reversion to more generalist pollination strategies.

  7. Transgressive phenotypes and generalist pollination in the floral evolution of Nicotiana polyploids.

    PubMed

    McCarthy, Elizabeth W; Chase, Mark W; Knapp, Sandra; Litt, Amy; Leitch, Andrew R; Le Comber, Steven C

    2016-01-01

    Polyploidy is an important driving force in angiosperm evolution, and much research has focused on genetic, epigenetic and transcriptomic responses to allopolyploidy. Nicotiana is an excellent system in which to study allopolyploidy because half of the species are allotetraploids of different ages, allowing us to examine the trajectory of floral evolution over time. Here, we study the effects of allopolyploidy on floral morphology in Nicotiana, using corolla tube measurements and geometric morphometrics to quantify petal shape. We show that polyploid morphological divergence from the intermediate phenotype expected (based on progenitor morphology) increases with time for floral limb shape and tube length, and that most polyploids are distinct or transgressive in at least one trait. In addition, we show that polyploids tend to evolve shorter and wider corolla tubes, suggesting that allopolyploidy could provide an escape from specialist pollination via reversion to more generalist pollination strategies. PMID:27501400

  8. Identification and validation of polymorphic microsatellite loci for the analysis of Phytophthora nicotianae populations.

    PubMed

    Biasi, Antonio; Martin, Frank; Schena, Leonardo

    2015-03-01

    A large number of SSR loci were screened in the genomic assemblies of 14 different isolates of Phytophthora nicotianae and primers were developed for amplification of 17 markers distributed among different contigs. These loci were highly polymorphic and amplified from genetically distant isolates of the pathogen. Among these, nine were further validated using a multiplexed genotyping assay with differentially labeled primers (FAM or HEX) to allow for duplex PCR amplification. The use of reverse primers with a 5' PIG tail was important to increase the quality and reliability of the analyses. A total of 46 alleles were detected in 5 tester isolates of P. nicotianae representing the breadth of diversity in the species. Furthermore, a high incidence of heterozygosity was determined with two alleles detected in 67% of the primer/isolate combinations. Three different alleles where detected for a single locus/isolate combination, indicating variation in ploidy. These markers represent a valuable new tool for the characterization of populations of P. nicotianae.

  9. The effect of polyploidy and hybridization on the evolution of floral colour in Nicotiana (Solanaceae)

    PubMed Central

    McCarthy, Elizabeth W.; Arnold, Sarah E. J.; Chittka, Lars; Le Comber, Steven C.; Verity, Robert; Dodsworth, Steven; Knapp, Sandra; Kelly, Laura J.; Chase, Mark W.; Baldwin, Ian T.; Kovařík, Aleš; Mhiri, Corinne; Taylor, Lin; Leitch, Andrew R.

    2015-01-01

    Background and Aims Speciation in angiosperms can be accompanied by changes in floral colour that may influence pollinator preference and reproductive isolation. This study investigates whether changes in floral colour can accompany polyploid and homoploid hybridization, important processes in angiosperm evolution. Methods Spectral reflectance of corolla tissue was examined for 60 Nicotiana (Solanaceae) accessions (41 taxa) based on spectral shape (corresponding to pigmentation) as well as bee and hummingbird colour perception in order to assess patterns of floral colour evolution. Polyploid and homoploid hybrid spectra were compared with those of their progenitors to evaluate whether hybridization has resulted in floral colour shifts. Key Results Floral colour categories in Nicotiana seem to have arisen multiple times independently during the evolution of the genus. Most younger polyploids displayed an unexpected floral colour, considering those of their progenitors, in the colour perception of at least one pollinator type, whereas older polyploids tended to resemble one or both of their progenitors. Conclusions Floral colour evolution in Nicotiana is weakly constrained by phylogeny, and colour shifts do occur in association with both polyploid and homoploid hybrid divergence. Transgressive floral colour in N. tabacum has arisen by inheritance of anthocyanin pigmentation from its paternal progenitor while having a plastid phenotype like its maternal progenitor. Potentially, floral colour evolution has been driven by, or resulted in, pollinator shifts. However, those polyploids that are not sympatric (on a regional scale) with their progenitor lineages are typically not divergent in floral colour from them, perhaps because of a lack of competition for pollinators. PMID:25979919

  10. Characterization of two cDNAs encoding auxin-binding proteins in Nicotiana tabacum.

    PubMed

    Leblanc, N; Roux, C; Pradier, J M; Perrot-Rechenmann, C

    1997-03-01

    The isolation and the characterization of two tobacco cDNAs, Nt-ERabp1 and Nt-ERabp2, homologous to Zm-ERabp1, encoding the major auxin-binding protein from maize coleoptiles, are described. Their predicted amino acid sequences correspond to proteins of ca. 21 kDa, in which the characteristic regions common to ABP1-related polypeptides are well-conserved. Southern analysis indicates that the genes corresponding to Nt-ERabp1 cDNA and Nt-ERabp2 cDNA derive respectively from Nicotiana tomentosiformis and Nicotiana sylvestris, the diploid progenitors of Nicotiana tabacum. Analysis of mRNA distribution in tobacco plants indicates that these two genes are preferentially expressed in flowers and growing seedlings. Whatever the tissue tested, Nt-ERabp1 mRNA is more abundant than Nt-ERabp2 mRNA. Furthermore, RT-PCR reveals developmental and organ-specific expression of these two genes in flower parts of tobacco plants. In particular, regulation of Nt-ERabp1 mRNA accumulation appears to be correlated with elongation growth of each floral organ. Recombinant Nt-ERabp1, produced in Escherichia coli, is recognized by antibodies raised against Zm-ERabp1.

  11. S-glycoprotein-like protein regulates defense responses in Nicotiana plants against Ralstonia solanacearum.

    PubMed

    Maimbo, Milimo; Ohnishi, Kouhei; Hikichi, Yasufumi; Yoshioka, Hirofumi; Kiba, Akinori

    2010-04-01

    RsRGA4 (for Ralstonia solanacearum-responsive gene A4) encodes a polypeptide similar to S-locus glycoprotein (SGP) from Brassica rapa and SGP-like proteins from Ipomoea trifida and Medicago truncatula. Therefore, we designated RsRGA4 as NtSGLP (for Nicotiana tabacum SGP-like protein) and NbSGLP (its Nicotiana benthamiana ortholog). NbSGLP is expressed in root, leaf, petal, gynoecium, and stamen. Expression of NbSGLP was strongly induced by inoculation with an avirulent strain of R. solanacearum (Rs8107) and slightly enhanced by inoculation with virulent R. solanacearum (RsOE1-1). Expression of NbSGLP was induced by inoculation with an hrpY-deficient mutant of RsOE1-1 and Rs8107. Expression was also induced by aminocyclopropane carboxylic acid and salicylic acid. Virus-induced gene silencing of NbSGLP enhanced the growth of Rs8107. Growth of RsOE1-1 and appearance of wilt symptoms were also accelerated in silenced plants. Expression of PR-1a and EREBP was reduced, and markers for basal defense, such as callose deposition and reduced vascular flow, were compromised in NbSGLP-silenced plants. Moreover, growth of Pseudomonas cichorii, Pseudomonas syringae pv tabaci, and P. syringae pv mellea was also enhanced in the silenced plants. On the other hand, silencing of NbSGLP did not interfere with the appearance of the hypersensitive response. NbSGLP was secreted in a signal peptide-dependent manner. Agrobacterium tumefaciens-mediated expression of NbSGLP induced PR-1a and EREBP expression, callose deposition, and reduced vascular flow. NbSGLP-induced callose deposition and reduced vascular flow were not observed in salicylic acid-deficient N. benthamiana NahG plants. Taken together, SGLP might have a role in the induction of basal defense in Nicotiana plants.

  12. Nicotiana Small RNA Sequences Support a Host Genome Origin of Cucumber Mosaic Virus Satellite RNA

    PubMed Central

    Smith, Neil A.; Schumann, Ulrike; Fang, Yuan-Yuan; Dennis, Elizabeth S.; Zhang, Ren; Guo, Hui-Shan; Wang, Ming-Bo

    2015-01-01

    Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs. PMID:25568943

  13. S-glycoprotein-like protein regulates defense responses in Nicotiana plants against Ralstonia solanacearum.

    PubMed

    Maimbo, Milimo; Ohnishi, Kouhei; Hikichi, Yasufumi; Yoshioka, Hirofumi; Kiba, Akinori

    2010-04-01

    RsRGA4 (for Ralstonia solanacearum-responsive gene A4) encodes a polypeptide similar to S-locus glycoprotein (SGP) from Brassica rapa and SGP-like proteins from Ipomoea trifida and Medicago truncatula. Therefore, we designated RsRGA4 as NtSGLP (for Nicotiana tabacum SGP-like protein) and NbSGLP (its Nicotiana benthamiana ortholog). NbSGLP is expressed in root, leaf, petal, gynoecium, and stamen. Expression of NbSGLP was strongly induced by inoculation with an avirulent strain of R. solanacearum (Rs8107) and slightly enhanced by inoculation with virulent R. solanacearum (RsOE1-1). Expression of NbSGLP was induced by inoculation with an hrpY-deficient mutant of RsOE1-1 and Rs8107. Expression was also induced by aminocyclopropane carboxylic acid and salicylic acid. Virus-induced gene silencing of NbSGLP enhanced the growth of Rs8107. Growth of RsOE1-1 and appearance of wilt symptoms were also accelerated in silenced plants. Expression of PR-1a and EREBP was reduced, and markers for basal defense, such as callose deposition and reduced vascular flow, were compromised in NbSGLP-silenced plants. Moreover, growth of Pseudomonas cichorii, Pseudomonas syringae pv tabaci, and P. syringae pv mellea was also enhanced in the silenced plants. On the other hand, silencing of NbSGLP did not interfere with the appearance of the hypersensitive response. NbSGLP was secreted in a signal peptide-dependent manner. Agrobacterium tumefaciens-mediated expression of NbSGLP induced PR-1a and EREBP expression, callose deposition, and reduced vascular flow. NbSGLP-induced callose deposition and reduced vascular flow were not observed in salicylic acid-deficient N. benthamiana NahG plants. Taken together, SGLP might have a role in the induction of basal defense in Nicotiana plants. PMID:20118275

  14. Influence of selected herbicides on ozone injury in tobacco (Nicotiana tabacum)

    SciTech Connect

    Reilly, J.J.; Moore, L.D.

    1982-01-01

    Field experiments were conducted over a four year period to determine the influence of selected herbicides on ozone injury in tobacco (Nicotiana tabacum L.). Isopropalin (2,6-dinitro-N,N-dipropylcumidine), pebulate (S-propyl butylethylthiocarbamate), and diphenamid (N,N-dimethyl-2,2-diphenylacetamide) were applied at the recommended rates of 1.7, 4.5, and 4.5 kg/ha (ai), respectively. Treatment of tobacco plants with isopropalin or diphenamid reduced oxidant injury for the first two to four weeks after transplanting, but not later in the season. Pebulate has no consistent affect on the sensitivity of tobacco to ozone. 26 references, 3 tables.

  15. Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum.

    PubMed

    Witters, Erwin; Quanten, Lieve; Bloemen, Jo; Valcke, Roland; Van Onckelen, Harry

    2004-01-01

    In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context.

  16. Specific expression of an extensin-like gene in the style of Nicotiana alata.

    PubMed Central

    Chen, C G; Cornish, E C; Clarke, A E

    1992-01-01

    cDNAs and corresponding genomic clones encoding a putative proline-rich protein (NaPRP3) were isolated from libraries prepared from Nicotiana alata style mRNA and genomic DNA. The predicted NaPRP3 protein is structurally similar to extensin in containing six copies of the characteristic extensin sequence Ser-Pro4, but differs in being smaller (151 residues compared with greater than 300 residues) and lacking Tyr residues. In contrast to most extensin genes, the NaPRP3 gene is not induced by mechanical wounding, and its expression is restricted to cells of the transmitting tract of the style. PMID:1392608

  17. [Distribution and environmental conditions related to the behavior in the bottlenose dolphin (Tursiops truncatus) and the spotted dolphin (Stenella attenuata) (Cetacea: Delphinidae) in Golfo Dulce, Costa Rica].

    PubMed

    Cubero-Pardo, Priscilla

    2007-06-01

    Habitat characteristics influencing behavior in animal species vary locally. The influence that a particular environmental characteristic can have on a species depends not only on other variables, but on morphological, physiological and social conditions of that species. In this study, developed from June 1996 to July 1997, I studied whether specific behaviors are related to particular distribution areas and environmental factors in the bottlenose (Tursiops truncatus) and the spotted dolphin (Stenella attenuata). The study area was covered along oblicuous linear transects, and the behavior of single groups was observed from 15 min to 5 h. Environmental factors such as depth, temperature, salinity and distance from shore, among others, were considered. For the bottlenose dolphin, foraging/feeding activities showed exclusive coincidence with river mouths, coral reef and mangrove areas, while social and milling activities where seen close to feeding areas. Traveling occurred along different points parallel to the coast, with a low percentage of cases across the gulf (16.56 %), suggesting that the bottlenose rarely crosses from one side to the other. In the spotted dolphin, several behaviors were observed simultaneously in the schools and it was not possible to associate areas with particular behaviors. The lack of significant relationships among activities and particular environmental variables (ANOVA tests) is attributed to three aspects: (a) transitions among activities generally occurred into a low variable area, (b) dolphins often traveled along large areas without changing activities and (c) environmental conditions in Golfo Dulce are homogeneous. In the two species the highest average in the number of individuals per group corresponded to the category of active socializing, followed by traveling, passive socializing and feeding. In the case of the bottlenose dolphin, the smallest group size was associated with feeding activities (ANOVA, F= 2.624, p=0.037, n=156

  18. Whistle characteristics and daytime dive behavior in pantropical spotted dolphins (Stenella attenuata) in Hawai'i measured using digital acoustic recording tags (DTAGs).

    PubMed

    Silva, Tammy L; Mooney, T Aran; Sayigh, Laela S; Tyack, Peter L; Baird, Robin W; Oswald, Julie N

    2016-07-01

    This study characterizes daytime acoustic and dive behavior of pantropical spotted dolphins (Stenella attenuata) in Hawai'i using 14.58 h of data collected from five deployments of digital acoustic recording tags (DTAG3) in 2013. For each tagged animal, the number of whistles, foraging buzzes, dive profiles, and dive statistics were calculated. Start, end, minimum, and maximum frequencies, number of inflection points and duration were measured from 746 whistles. Whistles ranged in frequency from 9.7 ± 2.8 to 19.8 ± 4.2 kHz, had a mean duration of 0.7 ± 0.5 s and a mean of 1.2 ± 1.2 inflection points. Thirteen foraging buzzes were recorded across all tags. Mean dive depth and duration were 16 ± 9 m and 1.9 ± 1.0 min, respectively. Tagged animals spent the majority of time in the upper 10 m (76.9% ± 16.1%) of the water column. Both whistle frequency characteristics and dive statistics measured here were similar to previously reported values for spotted dolphins in Hawai'i. Shallow, short dive profiles combined with few foraging buzzes provide evidence that little spotted dolphin feeding behavior occurs during daytime hours. This work represents one of the first successful DTAG3 studies of small pelagic delphinids, providing rare insights into baseline bioacoustics and dive behavior.

  19. Chloroplast DNA Diversity among Trees, Populations and Species in the California Closed-Cone Pines (Pinus Radiata, Pinus Muricata and Pinus Attenuata)

    PubMed Central

    Hong, Y. P.; Hipkins, V. D.; Strauss, S. H.

    1993-01-01

    The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% of the chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [G(st) = 84(+/-13)%]; nucleotide diversity among species was estimated to be 0.3(+/-0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [G(st) = 87(+/-8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/-0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga. PMID:7905846

  20. Whistle characteristics and daytime dive behavior in pantropical spotted dolphins (Stenella attenuata) in Hawai'i measured using digital acoustic recording tags (DTAGs).

    PubMed

    Silva, Tammy L; Mooney, T Aran; Sayigh, Laela S; Tyack, Peter L; Baird, Robin W; Oswald, Julie N

    2016-07-01

    This study characterizes daytime acoustic and dive behavior of pantropical spotted dolphins (Stenella attenuata) in Hawai'i using 14.58 h of data collected from five deployments of digital acoustic recording tags (DTAG3) in 2013. For each tagged animal, the number of whistles, foraging buzzes, dive profiles, and dive statistics were calculated. Start, end, minimum, and maximum frequencies, number of inflection points and duration were measured from 746 whistles. Whistles ranged in frequency from 9.7 ± 2.8 to 19.8 ± 4.2 kHz, had a mean duration of 0.7 ± 0.5 s and a mean of 1.2 ± 1.2 inflection points. Thirteen foraging buzzes were recorded across all tags. Mean dive depth and duration were 16 ± 9 m and 1.9 ± 1.0 min, respectively. Tagged animals spent the majority of time in the upper 10 m (76.9% ± 16.1%) of the water column. Both whistle frequency characteristics and dive statistics measured here were similar to previously reported values for spotted dolphins in Hawai'i. Shallow, short dive profiles combined with few foraging buzzes provide evidence that little spotted dolphin feeding behavior occurs during daytime hours. This work represents one of the first successful DTAG3 studies of small pelagic delphinids, providing rare insights into baseline bioacoustics and dive behavior. PMID:27475166

  1. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    PubMed Central

    Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2016-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  2. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  3. Chronic ozone exacerbates the reduction in photosynthesis and acceleration of senescence caused by limited N availability in Nicotiana sylvestris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elevated ozone (O3) and limiting soil nitrogen (N) availability both negatively affect crop performance. However, little is known about how the combination of elevated O3 and limiting N affect crop growth and metabolism. In this study, we grew tobacco (Nicotiana sylvestris) in ambient and elevated O...

  4. In vitro cytotoxicity of Nicotiana gossei leaves, used in the Australian Aboriginal smokeless tobacco known as pituri or mingkulpa.

    PubMed

    Moghbel, Nahid; Ryu, BoMi; Cabot, Peter J; Steadman, Kathryn J

    2016-07-01

    The Aboriginal population of Central Australia use endemic Nicotiana species to make a smokeless tobacco product known usually as pituri or mingkulpa. Nicotiana leaves are masticated with wood ash into a 'quid' that is chewed/sucked for absorption of nicotine. In addition to nicotine, smokeless tobacco products contain a spectrum of biologically active compounds that may contribute to effects on health. The objective of this study was to quantify nicotine, and related alkaloids and tobacco specific nitrosamines (TSNAs), in Nicotiana leaves used in pituri, and compare in vitro toxicity of pure nicotine with Nicotiana leaf extract at the same concentration of nicotine. An aqueous extract of dry leaves of Nicotiana gossei and a reference smokeless tobacco (CORESTA CRP2) were quantified for major pyridine alkaloids and TSNAs using HPLC-UV and LC-MS/MS. A range of extract concentrations and corresponding concentrations of nicotine standard were tested using an MTS assay to measure human lung epithelium cell (A549) survival. Cells treated for 24h with the maximum concentration of 1.5mg/ml of nicotine resulted in 77% viability. In contrast, extracts from N. gossei leaves and CRP2 containing a similar concentration of nicotine (1.3mg/ml) resulted in remarkably lower viability of 1.5 and 6%, respectively. Comparison of cytotoxicity of pure nicotine with that of the extracts revealed that nicotine was not the source of their cytotoxicity. Other biologically active compounds such as the known carcinogens NNK and NNN, derived from nicotine and nornicotine and found to be present in the smokeless tobacco extracts, may be responsible.

  5. In vitro cytotoxicity of Nicotiana gossei leaves, used in the Australian Aboriginal smokeless tobacco known as pituri or mingkulpa.

    PubMed

    Moghbel, Nahid; Ryu, BoMi; Cabot, Peter J; Steadman, Kathryn J

    2016-07-01

    The Aboriginal population of Central Australia use endemic Nicotiana species to make a smokeless tobacco product known usually as pituri or mingkulpa. Nicotiana leaves are masticated with wood ash into a 'quid' that is chewed/sucked for absorption of nicotine. In addition to nicotine, smokeless tobacco products contain a spectrum of biologically active compounds that may contribute to effects on health. The objective of this study was to quantify nicotine, and related alkaloids and tobacco specific nitrosamines (TSNAs), in Nicotiana leaves used in pituri, and compare in vitro toxicity of pure nicotine with Nicotiana leaf extract at the same concentration of nicotine. An aqueous extract of dry leaves of Nicotiana gossei and a reference smokeless tobacco (CORESTA CRP2) were quantified for major pyridine alkaloids and TSNAs using HPLC-UV and LC-MS/MS. A range of extract concentrations and corresponding concentrations of nicotine standard were tested using an MTS assay to measure human lung epithelium cell (A549) survival. Cells treated for 24h with the maximum concentration of 1.5mg/ml of nicotine resulted in 77% viability. In contrast, extracts from N. gossei leaves and CRP2 containing a similar concentration of nicotine (1.3mg/ml) resulted in remarkably lower viability of 1.5 and 6%, respectively. Comparison of cytotoxicity of pure nicotine with that of the extracts revealed that nicotine was not the source of their cytotoxicity. Other biologically active compounds such as the known carcinogens NNK and NNN, derived from nicotine and nornicotine and found to be present in the smokeless tobacco extracts, may be responsible. PMID:27178269

  6. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    PubMed

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

  7. Complete genome sequence of tobacco virus 1, a closterovirus from Nicotiana tabacum.

    PubMed

    Wang, Fang; Qi, Shuishui; Gao, Zhengliang; Akinyemi, Ibukun A; Xu, Dafeng; Zhou, Benguo

    2016-04-01

    The complete genome sequence of a novel virus, provisionally named tobacco virus 1 (TV1), was determined, and this virus was identified in leaves of tobacco (Nicotiana tabacum) exhibiting leaf mosaic and yellowing symptoms in Anhui Province, China. The genome sequence of TV1 consists of 15,395 nucleotides with 61.6 % nucleotide sequence identity to mint virus 1 (MV1). Its genome organization is similar to that of MV1, containing nine open reading frames (ORFs) that potentially encode proteins with putative functions in virion assembly, cell-to-cell movement and suppression of RNA silencing. Phylogenetic analysis of the heat shock protein 70 homolog (HSP70h) placed TV1 alongside members of the genus Closterovirus in the family Closteroviridae. To our knowledge, this study is the first report of the complete genome sequence of a closterovirus identified in tobacco. PMID:26795159

  8. The transcriptome of Verticillium dahliae-infected Nicotiana benthamiana determined by deep RNA sequencing.

    PubMed

    Faino, Luigi; de Jonge, Ronnie; Thomma, Bart P H J

    2012-09-01

    Verticillium wilt disease is caused by fungi of the Verticillium genus that occur on a wide range of host plants, including Solanaceous species such as tomato and tobacco. Currently, the well characterized Ve1 gene of tomato is the only Verticillium wilt resistance gene cloned. During experiments to identify the Verticillium molecule that activates Ve1 resistance in tomato, RNA sequencing (RNA-Seq) of Verticillium-infected Nicotiana benthamiana was performed. In total, over 99% of the obtained reads were derived from N. benthamiana. Here, we report the assembly and annotation of the N. benthamiana transcriptome. In total, 142,738 transcripts > 100 bp were obtained, amounting to a total transcriptome size of 38.7 Mbp, which is comparable to the Arabidopsis transcriptome. About 30,282 transcripts could be annotated based on homology to Arabidopsis genes. By assembly of the N. benthamiana transcriptome, we provide a catalogue of transcripts of a Solanaceous model plant under pathogen stress.

  9. Maternal synthesis of abscisic acid controls seed development and yield in Nicotiana plumbaginifolia.

    PubMed

    Frey, Anne; Godin, Béatrice; Bonnet, Magda; Sotta, Bruno; Marion-Poll, Annie

    2004-04-01

    The role of maternally derived abscisic acid (ABA) during seed development has been studied using ABA-deficient mutants of Nicotiana plumbaginifolia Viviani. ABA deficiency induced seed abortion, resulting in reduced seed yield, and delayed growth of the remaining embryos. Mutant grafting onto wild-type stocks and reciprocal crosses indicated that maternal ABA, synthesized in maternal vegetative tissues and translocated to the seed, promoted early seed development and growth. Moreover ABA deficiency delayed both seed coat pigmentation and capsule dehiscence. Mutant grafting did not restore these phenotypes, indicating that ABA synthesized in the seed coat and capsule envelope may have a positive effect on capsule and testa maturation. Together these results shed light on the positive role of maternal ABA during N. plumbaginifolia seed development.

  10. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    PubMed

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture. PMID:8470949

  11. Early events induced by the toxin deoxynivalenol lead to programmed cell death in Nicotiana tabacum cells.

    PubMed

    Yekkour, Amine; Tran, Daniel; Arbelet-Bonnin, Delphine; Briand, Joël; Mathieu, Florence; Lebrihi, Ahmed; Errakhi, Rafik; Sabaou, Nasserdine; Bouteau, François

    2015-09-01

    Deoxynivalenol (DON) is a mycotoxin affecting animals and plants. This toxin synthesized by Fusarium culmorum and Fusarium graminearum is currently believed to play a decisive role in the fungal phytopathogenesis as a virulence factor. Using cultured cells of Nicotiana tabacum BY2, we showed that DON-induced programmed cell death (PCD) could require transcription and translation processes, in contrast to what was observed in animal cells. DON could induce different cross-linked pathways involving (i) reactive oxygen species (ROS) generation linked, at least partly, to a mitochondrial dysfunction and a transcriptional down-regulation of the alternative oxidase (Aox1) gene and (ii) regulation of ion channel activities participating in cell shrinkage, to achieve PCD.

  12. Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana.

    PubMed

    Aslan, Selcuk; Sun, Chuanxin; Leonova, Svetlana; Dutta, Paresh; Dörmann, Peter; Domergue, Frédéric; Stymne, Sten; Hofvander, Per

    2014-09-01

    In a future bio-based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de novo fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl reductases (FAR) and wax ester synthases (WS) were transiently expressed in Nicotiana benthamiana leaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts.

  13. Metabolomic analysis of wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses: unraveling metabolic responses.

    PubMed

    Scalabrin, Elisa; Radaelli, Marta; Rizzato, Giovanni; Bogani, Patrizia; Buiatti, Marcello; Gambaro, Andrea; Capodaglio, Gabriele

    2015-08-01

    Nicotiana langsdorffii plants, wild and transgenic for the Agrobacterium rhizogenes rol C gene and the rat glucocorticoid receptor (GR) gene, were exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations). An untargeted metabolomic analysis was carried out in order to investigate the metabolic effects of the inserted genes in response to the applied stresses and to obtain a comprehensive profiling of metabolites induced during abiotic stresses. High-performance liquid chromatography separation (HPLC) coupled to high-resolution mass spectrometry (HRMS) enabled the identification of more than 200 metabolites, and statistical analysis highlighted the most relevant compounds for each plant treatment. The plants exposed to heat stress showed a unique set of induced secondary metabolites, some of which were known while others were not previously reported for this kind of stress; significant changes were observed especially in lipid composition. The role of trichome, as a protection against heat stress, is here suggested by the induction of both acylsugars and glykoalkaloids. Water deficit and Cr(VI) stresses resulted mainly in enhanced antioxidant (HCAs, polyamine) levels and in the damage of lipids, probably as a consequence of reactive oxygen species (ROS) production. Moreover, the ability of rol C expression to prevent oxidative burst was confirmed. The results highlighted a clear influence of GR modification on plant stress response, especially to water deficiency-a phenomenon whose applications should be further investigated. This study provides new insights into the field of system biology and demonstrates the importance of metabolomics in the study of plant functioning. Graphical Abstract Untargeted metabolomic analysis was applied to wild type, GR and RolC modified Nicotiana Langsdorffii plants exposed to heat, water and Cr(VI) stresses. The key metabolites, highly affected by stress application, were identified

  14. Regulation of catalase activity in leaves of Nicotiana sylvestris by high CO sub 2

    SciTech Connect

    Havir, E.A.; McHale, N.A. )

    1989-03-01

    The effect of high CO{sub 2} (1% CO{sub 2}/21% O{sub 2}) on the activity of specific forms of catalase (CAT-1, -2, and -3) in seedling leaves of tobacco (Nicotiana sylvestris, Nicotiana tabacum) was examined. In high CO{sub 2} total catalase activity decreased by 50% in the first 2 days, followed by a more gradual decline in the next 4 days. The loss of total activity resulted primarily from a decrease in CAT-1 catalase. In contrast, the activity of CAT-3 catalase, a form with enhanced peroxidatic activity, increased 3-fold in high CO{sub 2} relative to air controls after 4 days. Short-term exposure to high CO{sub 2} indicated that the 50% loss of total activity occurs in the firs 12 hours. Catalase levels increased to normal within 12 hours after seedlings were returned to air. When seedlings were transferred to air after prolonged exposure to high CO{sub 2} (13 days), the levels of CAT-1 catalase were partially restored while CAT-3 remained at its elevated level. Levels of superoxide dismutase activity and those of several peroxisomal enzymes were not affected by high CO{sub 2}. Total catalase levels did not decline when seedlings were exposed to atmospheres of 0.04% CO{sub 2}/5% O{sub 2} or 0.04% CO{sub 2}/1% O{sub 2}, indicating that regulation of catalase in high CO{sub 2} is not related directly to suppression of photorespiration. Antibodies prepared against CAT-1 catalase from N. tabacum reacted strongly against CAT-1 catalase from both N. sylvestris and N. tabacum but not against CAT-3 catalase from either species.

  15. Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

    PubMed

    Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J; Schaller, Hubert

    2014-01-01

    The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

  16. High-level diterpene production by transient expression in Nicotiana benthamiana

    PubMed Central

    2013-01-01

    Background Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis. Results Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2. Conclusion With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants. PMID:24330621

  17. Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in Nicotiana benthamiana

    PubMed Central

    Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J.; Schaller, Hubert

    2014-01-01

    The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ5-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis. PMID:25343375

  18. A functional genomics screen identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana.

    PubMed

    Todd, Andrea T; Liu, Enwu; Polvi, Sandra L; Pammett, Robert T; Page, Jonathan E

    2010-05-01

    Biosynthesis of the alkaloid nicotine in Nicotiana species is induced by insect damage and jasmonate application. To probe the transcriptional regulation of the nicotine pathway, we constructed two subtracted cDNA libraries from methyl jasmonate (MeJA)-treated Nicotiana benthamiana roots directly in a viral vector suitable for virus-induced gene silencing (VIGS). Sequencing of cDNA inserts produced a data set of 3271 expressed sequence tags (ESTs; 1898 unigenes), which were enriched in jasmonate-responsive genes, and included 69 putative transcription factors (TFs). After a VIGS screen to determine their effect on nicotine metabolism, six TFs from three different TF families altered constitutive and MeJA-induced leaf nicotine levels. VIGS of a basic helix-loop-helix (bHLH) TF, NbbHLH3, and an auxin response factor TF, NbARF1, increased nicotine content compared with control plants; silencing the bHLH family members, NbbHLH1 and NbbHLH2, an ethylene response factor TF, NbERF1, and a homeobox domain-like TF, NbHB1, reduced nicotine levels. Transgenic N. benthamiana plants overexpressing NbbHLH1 or NbbHLH2 showed increased leaf nicotine levels compared with vector controls. RNAi silencing led to both reduced nicotine and decreased levels of transcript encoding of enzymes of the nicotine pathway. Electrophoretic mobility shift assays showed that recombinant NbbHLH1 and NbbHLH2 directly bind G-box elements identified from the putrescine N-methyltransferase promoter. We conclude that NbbHLH1 and NbbHLH2 function as positive regulators in the jasmonate activation of nicotine biosynthesis. PMID:20202168

  19. Metabolomic analysis of wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses: unraveling metabolic responses.

    PubMed

    Scalabrin, Elisa; Radaelli, Marta; Rizzato, Giovanni; Bogani, Patrizia; Buiatti, Marcello; Gambaro, Andrea; Capodaglio, Gabriele

    2015-08-01

    Nicotiana langsdorffii plants, wild and transgenic for the Agrobacterium rhizogenes rol C gene and the rat glucocorticoid receptor (GR) gene, were exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations). An untargeted metabolomic analysis was carried out in order to investigate the metabolic effects of the inserted genes in response to the applied stresses and to obtain a comprehensive profiling of metabolites induced during abiotic stresses. High-performance liquid chromatography separation (HPLC) coupled to high-resolution mass spectrometry (HRMS) enabled the identification of more than 200 metabolites, and statistical analysis highlighted the most relevant compounds for each plant treatment. The plants exposed to heat stress showed a unique set of induced secondary metabolites, some of which were known while others were not previously reported for this kind of stress; significant changes were observed especially in lipid composition. The role of trichome, as a protection against heat stress, is here suggested by the induction of both acylsugars and glykoalkaloids. Water deficit and Cr(VI) stresses resulted mainly in enhanced antioxidant (HCAs, polyamine) levels and in the damage of lipids, probably as a consequence of reactive oxygen species (ROS) production. Moreover, the ability of rol C expression to prevent oxidative burst was confirmed. The results highlighted a clear influence of GR modification on plant stress response, especially to water deficiency-a phenomenon whose applications should be further investigated. This study provides new insights into the field of system biology and demonstrates the importance of metabolomics in the study of plant functioning. Graphical Abstract Untargeted metabolomic analysis was applied to wild type, GR and RolC modified Nicotiana Langsdorffii plants exposed to heat, water and Cr(VI) stresses. The key metabolites, highly affected by stress application, were identified

  20. Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells.

    PubMed

    Vatsa, Parul; Chiltz, Annick; Luini, Estelle; Vandelle, Elodie; Pugin, Alain; Roblin, Gabriel

    2011-07-01

    The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca²⁺](cyt) which intensity depends on ergosterol concentration in the range 0.002-10 μM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterol-induced pH and [Ca²⁺](cyt) changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl₃, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca²⁺](cyt) increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calcium-independent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus.

  1. Simultaneous Detection and Quantification of Phytophthora nicotianae and P. cactorum, and Distribution Analyses in Strawberry Greenhouses by Duplex Real-time PCR

    PubMed Central

    Li, Mingzhu; Inada, Minoru; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

    2013-01-01

    Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)−1. In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)−1 for P. nicotianae and from 13 to 233 pg (g soil)−1 for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils. PMID:23614901

  2. Simultaneous detection and quantification of Phytophthora nicotianae and P. cactorum, and distribution analyses in strawberry greenhouses by duplex real-time PCR.

    PubMed

    Li, Mingzhu; Inada, Minoru; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

    2013-01-01

    Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.

  3. An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

    PubMed Central

    Ling, Huai-Yian; Edwards, Aaron M.; Gantier, Michael P.; DeBoer, Kathleen D.; Neale, Alan D.; Hamill, John D.; Walmsley, Amanda M.

    2012-01-01

    The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs. PMID:22539991

  4. Enzymatic, expression and structural divergences among carboxyl O-methyltransferases after gene duplication and speciation in Nicotiana.

    PubMed

    Hippauf, Frank; Michalsky, Elke; Huang, Ruiqi; Preissner, Robert; Barkman, Todd J; Piechulla, Birgit

    2010-02-01

    Methyl salicylate and methyl benzoate have important roles in a variety of processes including pollinator attraction and plant defence. These compounds are synthesized by salicylic acid, benzoic acid and benzoic acid/salicylic acid carboxyl methyltransferases (SAMT, BAMT and BSMT) which are members of the SABATH gene family. Both SAMT and BSMT were isolated from Nicotiana suaveolens, Nicotiana alata, and Nicotiana sylvestris allowing us to discern levels of enzyme divergence resulting from gene duplication in addition to species divergence. Phylogenetic analyses showed that Nicotiana SAMTs and BSMTs evolved in separate clades and the latter can be differentiated into the BSMT1 and the newly established BSMT2 branch. Although SAMT and BSMT orthologs showed minimal change coincident with species divergences, substantial evolutionary change of enzyme activity and expression patterns occurred following gene duplication. After duplication, the BSMT enzymes evolved higher preference for benzoic acid (BA) than salicylic acid (SA) whereas SAMTs maintained ancestral enzymatic preference for SA over BA. Expression patterns are largely complementary in that BSMT transcripts primarily accumulate in flowers, leaves and stems whereas SAMT is expressed mostly in roots. A novel enzyme, nicotinic acid carboxyl methyltransferase (NAMT), which displays a high degree of activity with nicotinic acid was discovered to have evolved in N. gossei from an ancestral BSMT. Furthermore a SAM-dependent synthesis of methyl anthranilate via BSMT2 is reported and contrasts with alternative biosynthetic routes previously proposed. While BSMT in flowers is clearly involved in methyl benzoate synthesis to attract pollinators, its function in other organs and tissues remains obscure.

  5. Characterization and phylogenetic analysis of fifteen NtabSPL genes in Nicotiana tabacum L. cv. Qinyan95.

    PubMed

    Han, Yao-Yao; Ma, Yan-Qin; Li, Dian-Zhen; Yao, Jing-Wen; Xu, Zi-Qin

    2016-01-01

    Fifteen SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) genes were identified and characterized in Nicotiana tabacum L. cv. Qinyan95. The exon-intron structures of these genes were determined according to the coding sequences confirmed by RT-PCR and the genomic DNA sequences downloaded from the databases in Sol Genomics Network, and thirteen of them were found to carry the response element of miR156. To elucidate the origin of the validated NtabSPL genes, multiple alignments of the nucleotide sequences encompassing the open reading frames were conducted by using the orthologs in N. tabacum, Nicotiana sylvestris, Nicotiana tomentosiformis, and Nicotiana otophora. The results showed that six NtabSPL genes were derived from a progenitor of N. sylvestris, and nine NtabSPL genes were derived from a progenitor of N. tomentosiformis, further corroborating that N. tabacum came from the interspecific hybridization between the ancestors of N. sylvestris and N. tomentosiformis. In contrast to previous statements about highly repetitive sequences, the genome of N. tabacum mainly retained the paternal-derived SPL genes in diploidization process. Phylogenetic analyses based on the highly conserved SBP (SQUAMOSA PROMOTER BINDING PROTEIN) domains and the full-length amino acid sequences reveal that the SPL proteins of tobacco, tomato, and Arabidopsis can be categorized into eight groups. It is worth noting that N. tabacum contains seven NtabSPL6 genes originated from two parental genomes and NtabSPL6-2 possesses a GC-AG intron. In addition, transgenic tobacco plants harboring Arabidopsis Pri-miR156A were generated by Agrobacterium-mediated transformation method, and the constitutive expression of miR156 could obviously inhibit the activity of the NtabSPL genes containing its target site, suggesting the function of miR156 is conservative in tobacco and Arabidopsis.

  6. Visual monitoring of Cucumber mosaic virus infection in Nicotiana benthamiana following transmission by the aphid vector Myzus persicae.

    PubMed

    Krenz, Bjoern; Bronikowski, Agathe; Lu, Xiaoyun; Ziebell, Heiko; Thompson, Jeremy R; Perry, Keith L

    2015-09-01

    The single-stranded, positive-sense and tripartite RNA virus Cucumber mosaic virus (CMV) was used in this study as a method for monitoring the initial stages of virus infection following aphid transmission. The RNA2 of CMV was modified to incorporate, in a variety of arrangements, an open reading frame (ORF) encoding an enhanced green fluorescent protein (eGFP). The phenotypes of five engineered RNA2s were tested in Nicotiana tabacum, Nicotiana clevelandii and Nicotiana benthamiana. Only one construct (F4), in which the 2b ORF was truncated at the 3' end and fused in-frame with the eGFP ORF, was able to systemically infect N. benthamiana plants, express eGFP and be transmitted by the aphid Myzus persicae. The utility of this construct was demonstrated following infection as early as one day post-transmission (dpt) continuing through to systemic infection. Comparisons of the inoculation sites in different petiole sections one to three dpt clearly showed that the onset of infection and eGFP expression always occurred in the epidermal or collenchymatous tissue just below the epidermis; an observation consistent with the rapid time frame characteristic of the non-persistent mode of aphid transmission. PMID:25979730

  7. Comparison of cleft palate induction by Nicotiana glauca in goats and sheep.

    PubMed

    Panter, K E; Weinzweig, J; Gardner, D R; Stegelmeier, B L; James, L F

    2000-03-01

    The induction of cleft palate by Nicotiana glauca (wild tree tobacco) during the first trimester of pregnancy was compared between Spanish-type goats and crossbred western-type sheep. Cleft palate was induced in 100% of the embryonic/fetal goats when their pregnant mothers were gavaged with N. glauca plant material or with anabasine-rich extracts from the latter, during gestation days 32-41. Seventy-five percent of newborn goats had cleft palate after maternal dosing with N. glauca during gestation days 35-41, while no cleft palates were induced when dosing periods included days 36-40, 37-39, or day 38 only. The induced cleft palates were bilateral, involving the entire secondary palates with complete detachment of the vomer. Eleven percent of the newborn goats from does gavaged during gestation days 32-41 had extracranial abnormalities, most often contractures of the metacarpal joints. Most of these contractures resolved spontaneously by 4-6 weeks postpartum. One newborn kid also had an asymmetric skull due to apparent fetal positioning. No cleft palates were induced in lambs whose mothers were gavaged with N. glauca plant or anabasine-rich extracts during gestation days 34-41, 35-40, 35-41, 36-41, 35-51, or 37-50. Only one of five lambs born to three ewes gavaged with N. glauca plant material during gestation days 34-55 had a cleft palate, but all five of these lambs had moderate to severe contractures in the metacarpal joints. The slight to moderate contracture defects resolved spontaneously by 4-6 weeks postpartum, but the severe contractures resolved only partially. Embryonic/fetal death and resorption (determined by ultrasound) occurred in 25% of pregnant goats fed N. glauca compared to only 4% of pregnant sheep. Nicotiana glauca plant material contained the teratogenic alkaloid anabasine at 0.175% to 0.23%, dry weight, demonstrating that Spanish-type goats are susceptible to cleft palate induction by the natural toxin anabasine, while crossbred western

  8. Light and clomazone effects on tobacco (Nicotiana tabacum) callus and leaf discs.

    PubMed

    Camper, N D; McDonald, S K; Burrows, P M

    2003-11-01

    The effects of clomazone on the growth of tobacco (Nicotiana tabacum L. 'NC2326') callus and leaf discs were studied under four light regimes. Callus cultures and leaf discs were grown on Murashige and Skoog medium supplemented with IAA and kinetin. Light regimes were: dark grown callus kept in the dark and also transferred to the light; light grown callus kept in the light and also transferred to the dark. Two-month-old callus (cultured for 2 months from initiation) grew more rapidly than twelve-month-old callus (cultured for 12 months from initiation) under all conditions tested. Callus transferred from light to dark, or from dark to light, increased in fresh weight slower than did the callus maintained totally in light or dark. Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) at 140 mg l(-1) or more was lethal to both callus and leaf discs whereas 10 mg l(-1) was stimulatory to growth. Callus tissue responded to clomazone differently depending on the light regime under which it was grown. While clomazone may be affecting the isoprenoid pathway in the callus and leaf disks resulting in growth inhibition, it is possible that other target sites are also being affected and contribute to the reduced growth.

  9. GhWRKY68 Reduces Resistance to Salt and Drought in Transgenic Nicotiana benthamiana

    PubMed Central

    Jia, Haihong; Wang, Chen; Wang, Fang; Liu, Shuchang; Li, Guilin; Guo, Xingqi

    2015-01-01

    The WRKY transcription factors modulate numerous physiological processes, including plant growth, development and responses to various environmental stresses. Currently, our understanding of the functions of the majority of the WRKY family members and their possible roles in signalling crosstalk is limited. In particular, very few WRKYs have been identified and characterised from an economically important crop, cotton. In this study, we characterised a novel group IIc WRKY gene, GhWRKY68, which is induced by different abiotic stresses and multiple defence-related signalling molecules. The β-glucuronidase activity driven by the GhWRKY68 promoter was enhanced after exposure to drought, salt, abscisic acid (ABA) and H2O2. The overexpression of GhWRKY68 in Nicotiana benthamiana reduced resistance to drought and salt and affected several physiological indices. GhWRKY68 may mediate salt and drought responses by modulating ABA content and enhancing the transcript levels of ABA-responsive genes. GhWRKY68-overexpressing plants exhibited reduced tolerance to oxidative stress after drought and salt stress treatments, which correlated with the accumulation of reactive oxygen species (ROS), reduced enzyme activities, elevated malondialdehyde (MDA) content and altered ROS-related gene expression. These results indicate that GhWRKY68 is a transcription factor that responds to drought and salt stresses by regulating ABA signalling and modulating cellular ROS. PMID:25793865

  10. Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor.

    PubMed

    Diensthuber, Ralph P; Tominaga, Motoki; Preller, Matthias; Hartmann, Falk K; Orii, Hidefumi; Chizhov, Igor; Oiwa, Kazuhiro; Tsiavaliaris, Georgios

    2015-01-01

    The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a high duty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins. PMID:25326536

  11. Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum.

    PubMed

    Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

    2015-01-01

    Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand (TRAIL) as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV (Cauliflower Mosaic Virus) helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay (Methylthiazol Tetrazolium Assay). The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable.

  12. Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi

    PubMed Central

    Gore, Nigel R.; Wray, John L.

    1978-01-01

    Leucine:tRNA ligase was assayed in extracts from cultured tobacco (Nicotiana tabacum) XD cells by measuring the initial rate of aminoacylation of transfer RNA with l-[4,5-3H]leucine. Transfer RNA was purified from tobacco XD cells after the method of Vanderhoef et al. (Phytochemistry 9: 2291-2304). The buoyant density of leucine:tRNA ligase from cells grown for 100 generations in 2.5 mm [15N]nitrate and 30% deuterium oxide was 1.3397. After transfer of cells into light medium (2.5 mm [14N]nitrate and 100% H2O) the ligase activity increased and the buoyant density decreased with time to 1.3174 at 72 hours after transfer. It was concluded that leucine:tRNA ligase molecules were synthesized de novo from light amino acids during the period of activity increase. The width at half-peak height of the enzyme distribution profiles following isopycnic equilibrium centrifugation in caesium chloride remained constant at all times after transfer into light medium providing evidence for the loss of preexisting functional ligase molecules. It was concluded that during the period of activity increase the cellular level of enzyme activity was determined by a balance between de novo synthesis and the loss of functional enzyme molecules due to either inactivation or degradation. PMID:16660229

  13. Influence of Iron Chlorosis on Pigment and Protein Metabolism in Leaves of Nicotiana tabacum L. 1

    PubMed Central

    Shetty, A. S.; Miller, G. W.

    1966-01-01

    Experiments were conducted on Nicotiana tabacum, L. to study the relation in the grana among chlorophylls, carotenoids, and proteins. The effect of iron chlorosis on protein and pigment synthesis was studied at different stages of chlorosis using glycine-U-C14. Pigments were separated by thin layer chromatography. Chlorophyll a, chlorophyll b, carotenoid, and protein contents of chloroplasts from chlorotic tissue were less than those of normal tissues. A 25% decrease in protein labeling and a 45% decrease in chlorophyll labeling was noted in deficient tissue compared to normal tissue even before chlorosis was perceptible. Both normal and iron deficient leaf discs which received iron in the incubation medium incorporated higher amounts of radioactive glycine into chlorophyll a and chlorophyll b at all stages of development than their respective counterparts not supplied with iron in the incubation medium. The presence of iron in the incubation medium reduced the amount of glycine incorporated into carotenes and xanthophylls, except where the tissue was severely chlorotic. This may be attributed to active competition for glycine between the iron-dependent- (chlorophyll) and iron-independent-(carotenoid) biosynthetic pathways. Incorporation of glycine into chloroplast pigments was lowest at severe chlorosis, probably due to a reduction in the overall enzyme activity. PMID:16656270

  14. Mitochondrial alternative oxidase maintains respiration and preserves photosynthetic capacity during moderate drought in Nicotiana tabacum.

    PubMed

    Dahal, Keshav; Wang, Jia; Martyn, Greg D; Rahimy, Farkhunda; Vanlerberghe, Greg C

    2014-11-01

    The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum 'Petit Havana SR1' wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate drought-stressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO2 and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity.

  15. GhWRKY68 reduces resistance to salt and drought in transgenic Nicotiana benthamiana.

    PubMed

    Jia, Haihong; Wang, Chen; Wang, Fang; Liu, Shuchang; Li, Guilin; Guo, Xingqi

    2015-01-01

    The WRKY transcription factors modulate numerous physiological processes, including plant growth, development and responses to various environmental stresses. Currently, our understanding of the functions of the majority of the WRKY family members and their possible roles in signalling crosstalk is limited. In particular, very few WRKYs have been identified and characterised from an economically important crop, cotton. In this study, we characterised a novel group IIc WRKY gene, GhWRKY68, which is induced by different abiotic stresses and multiple defence-related signalling molecules. The β-glucuronidase activity driven by the GhWRKY68 promoter was enhanced after exposure to drought, salt, abscisic acid (ABA) and H2O2. The overexpression of GhWRKY68 in Nicotiana benthamiana reduced resistance to drought and salt and affected several physiological indices. GhWRKY68 may mediate salt and drought responses by modulating ABA content and enhancing the transcript levels of ABA-responsive genes. GhWRKY68-overexpressing plants exhibited reduced tolerance to oxidative stress after drought and salt stress treatments, which correlated with the accumulation of reactive oxygen species (ROS), reduced enzyme activities, elevated malondialdehyde (MDA) content and altered ROS-related gene expression. These results indicate that GhWRKY68 is a transcription factor that responds to drought and salt stresses by regulating ABA signalling and modulating cellular ROS. PMID:25793865

  16. Reduced Susceptibility to Xanthomonas citri in Transgenic Citrus Expressing the FLS2 Receptor From Nicotiana benthamiana.

    PubMed

    Hao, Guixia; Pitino, Marco; Duan, Yongping; Stover, Ed

    2016-02-01

    Overexpression of plant pattern-recognition receptors by genetic engineering provides a novel approach to enhance plant immunity and broad-spectrum disease resistance. Citrus canker disease associated with Xanthomonas citri is one of the most important diseases damaging citrus production worldwide. In this study, we cloned the FLS2 gene from Nicotiana benthamiana cDNA and inserted it into the binary vector pBinPlus/ARS to transform Hamlin sweet orange and Carrizo citrange. Transgene presence was confirmed by polymerase chain reaction (PCR) and gene expression of NbFLS2 was compared by reverse transcription quantitative PCR. Reactive oxygen species (ROS) production in response to flg22Xcc was detected in transgenic Hamlin but not in nontransformed controls. Low or no ROS production was detected from nontransformed Hamlin seedlings challenged with flg22Xcc. Transgenic plants highly expressing NbFLS2 were selected and were evaluated for resistance to canker incited by X. citri 3213. Our results showed that the integration and expression of the NbFLS2 gene in citrus can increase canker resistance and defense-associated gene expression when challenged with X. citri. These results suggest that canker-susceptible Citrus genotypes lack strong basal defense induced by X. citri flagellin and the resistance of these genotypes can be enhanced by transgenic expression of the flagellin receptor from a resistant species. PMID:26554734

  17. In vivo effects of NbSiR silencing on chloroplast development in Nicotiana benthamiana.

    PubMed

    Kang, Yong-Won; Lee, Jae-Yong; Jeon, Young; Cheong, Gang-Won; Kim, Moonil; Pai, Hyun-Sook

    2010-04-01

    Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development. PMID:20047069

  18. S1 domain-containing STF modulates plastid transcription and chloroplast biogenesis in Nicotiana benthamiana.

    PubMed

    Jeon, Young; Jung, Hyun Ju; Kang, Hunseung; Park, Youn-Il; Lee, Soon Hee; Pai, Hyun-Sook

    2012-01-01

    • In this study, we examined the biochemical and physiological functions of Nicotiana benthamiana S1 domain-containing Transcription-Stimulating Factor (STF) using virus-induced gene silencing (VIGS), cosuppression, and overexpression strategies. • STF : green fluorescent protein (GFP) fusion protein colocalized with sulfite reductase (SiR), a chloroplast nucleoid-associated protein also present in the stroma. Full-length STF and its S1 domain preferentially bound to RNA, probably in a sequence-nonspecific manner. • STF silencing by VIGS or cosuppression resulted in severe leaf yellowing caused by disrupted chloroplast development. STF deficiency significantly perturbed plastid-encoded multimeric RNA polymerase (PEP)-dependent transcript accumulation. Chloroplast transcription run-on assays revealed that the transcription rate of PEP-dependent plastid genes was reduced in the STF-silenced leaves. Conversely, the exogenously added recombinant STF protein increased the transcription rate, suggesting a direct role of STF in plastid transcription. Etiolated seedlings of STF cosuppression lines showed defects in the light-triggered transition from etioplasts to chloroplasts, accompanied by reduced light-induced expression of plastid-encoded genes. • These results suggest that STF plays a critical role as an auxiliary factor of the PEP transcription complex in the regulation of plastid transcription and chloroplast biogenesis in higher plants. PMID:22050604

  19. Molecular cloning and functional characterization of a putative sulfite oxidase (SO) ortholog from Nicotiana benthamiana.

    PubMed

    Xia, Zongliang; Su, Xinhong; Wu, Jianyu; Wu, Ke; Zhang, Hua

    2012-03-01

    Sulfite oxidase (SO) catalyzes the oxidation of sulfite to sulfate and thus has important roles in diverse metabolic processes. However, systematic molecular and functional investigations on the putative SO from tobacco (Nicotiana benthamiana) have hitherto not been reported. In this work, a full-length cDNA encoding putative sulfite oxidase from N. benthamiana (NbSO) was isolated. The deduced NbSO protein shares high homology and typical structural features with other species SOs. Phylogenetic analysis indicates that NbSO cDNA clone encodes a tobacco SO isoform. Southern blot analysis suggests that NbSO is a single-copy gene in the N. benthamiana genome. The NbSO transcript levels were higher in aerial tissues and were up-regulated in N. benthamiana during sulfite stress. Reducing the SO expression levels through virus-induced gene silencing caused a substantial accumulation in sulfite content and less sulfate accumulation in N. benthamiana leaves when exposed to sulfite stress, and thus resulted in decreased tolerance to sulfite stress. Taken together, this study improves our understanding on the molecular and functional properties of plant SO and provides genetic evidence on the involvement of SO in sulfite detoxification in a sulfite-oxidizing manner in N. benthamiana plants. PMID:21667106

  20. WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana.

    PubMed

    Adachi, Hiroaki; Nakano, Takaaki; Miyagawa, Noriko; Ishihama, Nobuaki; Yoshioka, Miki; Katou, Yuri; Yaeno, Takashi; Shirasu, Ken; Yoshioka, Hirofumi

    2015-09-01

    Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB. PMID:26373453

  1. Identification of Multivesicular Bodies as Prevacuolar Compartments in Nicotiana tabacum BY-2 CellsW⃞

    PubMed Central

    Tse, Yu Chung; Mo, Beixin; Hillmer, Stefan; Zhao, Min; Lo, Sze Wan; Robinson, David G.; Jiang, Liwen

    2004-01-01

    Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs). We have demonstrated recently that vacuolar sorting receptor (VSR) proteins are concentrated on PVCs. In this study, we generated transgenic Nicotiana tabacum (tobacco) BY-2 cell lines expressing two yellow fluorescent protein (YFP)-fusion reporters that mark PVC and Golgi organelles. Both transgenic cell lines exhibited typical punctate YFP signals corresponding to distinct PVC and Golgi organelles because the PVC reporter colocalized with VSR proteins, whereas the Golgi marker colocalized with mannosidase I in confocal immunofluorescence. Brefeldin A induced the YFP-labeled Golgi stacks but not the YFP-marked PVCs to form typical enlarged structures. By contrast, wortmannin caused YFP-labeled PVCs but not YFP-labeled Golgi stacks to vacuolate. VSR antibodies labeled multivesicular bodies (MVBs) on thin sections prepared from high-pressure frozen/freeze substituted samples, and the enlarged PVCs also were indentified as MVBs. MVBs were further purified from BY-2 cells and found to contain VSR proteins via immunogold negative staining. Similar to YFP-labeled Golgi stacks, YFP-labeled PVCs are mobile organelles in BY-2 cells. Thus, we have unequivocally identified MVBs as PVCs in N. tabacum BY-2 cells. Uptake studies with the styryl dye FM4-64 strongly indicate that PVCs also lie on the endocytic pathway of BY-2 cells. PMID:14973159

  2. The extremophile Nicotiana benthamiana has traded viral defence for early vigour.

    PubMed

    Bally, Julia; Nakasugi, Kenlee; Jia, Fangzhi; Jung, Hyungtaek; Ho, Simon Y W; Wong, Mei; Paul, Chloe M; Naim, Fatima; Wood, Craig C; Crowhurst, Ross N; Hellens, Roger P; Dale, James L; Waterhouse, Peter M

    2015-01-01

    A single lineage of Nicotiana benthamiana is widely used as a model plant(1) and has been instrumental in making revolutionary discoveries about RNA interference (RNAi), viral defence and vaccine production. It is peerless in its susceptibility to viruses and its amenability in transiently expressing transgenes(2,3). These unparalleled characteristics have been associated both positively and negatively with a disruptive insertion in the RNA-dependent RNA polymerase 1 gene, Rdr1(4-6). For a plant so routinely used in research, the origin, diversity and evolution of the species, and the basis of its unusual abilities, have been relatively unexplored. Here, by comparison with wild accessions from across the spectrum of the species' natural distribution, we show that the laboratory strain of N. benthamiana is an extremophile originating from a population that has retained a mutation in Rdr1 for ∼0.8 Myr and thereby traded its defence capacity for early vigour and survival in the extreme habitat of central Australia. Reconstituting Rdr1 activity in this isolate provided protection. Silencing the functional allele in a wild strain rendered it hypersusceptible and was associated with a doubling of seed size and enhanced early growth rate. These findings open the way to a deeper understanding of the delicate balance between protection and vigour. PMID:27251536

  3. Replication-independent long-distance trafficking by viral RNAs in Nicotiana benthamiana.

    PubMed

    Gopinath, Kodetham; Kao, C Cheng

    2007-04-01

    Viruses with separately encapsidated genomes could have their genomes introduced into different leaves of a plant, thus necessitating long-distance trafficking of the viral RNAs for successful infection. To examine this possibility, individual or combinations of genome segments from the tripartite Brome mosaic virus (BMV) were transiently expressed in leaves of Nicotiana benthamiana plants using engineered Agrobacterium tumefaciens. BMV RNA3 was found to traffic from the initial site of expression to other leaves of the plant, as detected by RNA gel blot analyses and also by the expression of an endoplasmic reticulum-targeted green fluorescent protein. When RNA3 trafficked into leaves containing the BMV replication enzymes, RNA replication, transcription, and virion production were observed. RNA3 trafficking occurred even when it did not encode the movement or capsid proteins. However, coexpression of the movement protein increased the trafficking of BMV RNAs. BMV RNA1 and RNA2 could also traffic throughout the plant, but less efficiently than RNA3. All three BMV RNAs trafficked bidirectionally to sink leaves near the apical meristem as well as to the source leaves at the bottom of the stem, suggesting that trafficking used the phloem. These results demonstrate that BMV RNAs can use a replication-independent mechanism to traffic in N. benthamiana.

  4. Pathway for the biosynthesis of 4-methyl-1-hexanol volatilized from petal tissue of Nicotiana sylvestris.

    PubMed

    Kandra, Lili; Wagner, George J.

    1998-11-20

    Compounds volatilized from plant tissues play important roles in plant-insect and plant-herbivore interactions and are important to food quality/preference, and to the perfume and flavorant industries. While the chemistry of plant volatiles is well understood, less is known about the biosynthesis of this diverse group of compounds. This is particularly the case for non-terpenoid components such as volatile acyclic alcohols and their esters. Here we have studied metabolic pathways leading to formation of the anteiso-branched alcohol 4-methyl-1-hexanol volatilized by petal tissue of Nicotiana sylvestris. Evidence presented supports the involvement of steps in the pathways of both biosynthesis and degradation of isoleucine to form 2-oxo-3-methylvaleric acid then 2-methylbutyryl CoA. Results indicate that 2-methylbutyryl CoA is then elongated by addition of one acetate molecule via fatty acid synthase, followed by reduction to yield 4-methyl-1-hexanol. This pathway is in contrast to elongation of 2-oxo-3-methylvaleric acid via alpha-keto acid elongation leading to the formation of 4-methylhexanoyl acyl groups of tobacco leaf-trichome-secreted sugar esters.

  5. Transient fusion and selective secretion of vesicle proteins in Phytophthora nicotianae zoospores

    PubMed Central

    Zhang, Weiwei; Blackman, Leila M.

    2013-01-01

    Secretion of pathogen proteins is crucial for the establishment of disease in animals and plants. Typically, early interactions between host and pathogen trigger regulated secretion of pathogenicity factors that function in pathogen adhesion and host penetration. During the onset of plant infection by spores of the Oomycete, Phytophthora nicotianae, proteins are secreted from three types of cortical vesicles. Following induction of spore encystment, two vesicle types undergo full fusion, releasing their entire contents onto the cell surface. However, the third vesicle type, so-called large peripheral vesicles, selectively secretes a small Sushi domain-containing protein, PnCcp, while retaining a large glycoprotein, PnLpv, before moving away from the plasma membrane. Selective secretion of PnCcp is associated with its compartmentalization within the vesicle periphery. Pharmacological inhibition of dynamin function, purportedly in vesicle fission, by dynasore treatment provides evidence that selective secretion of PnCcp requires transient fusion of the large peripheral vesicles. This is the first report of selective protein secretion via transient fusion outside mammalian cells. Selective secretion is likely to be an important aspect of plant infection by this destructive pathogen. PMID:24392285

  6. Improved photosynthetic performance during severe drought in Nicotiana tabacum overexpressing a nonenergy conserving respiratory electron sink.

    PubMed

    Dahal, Keshav; Martyn, Greg D; Vanlerberghe, Greg C

    2015-10-01

    Chloroplasts have means to manage excess reducing power but these mechanisms may become restricted by rates of ATP turnover. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that uncouples the consumption of reducing power from ATP synthesis. Physiological and biochemical analyses were used to compare respiration and photosynthesis of Nicotiana tabacum wild-type (WT) plants with that of transgenic lines overexpressing AOX, under both well-watered and drought stress conditions. With increasing drought severity, AOX overexpression acted to increase respiration in the light (RL ) relative to WT. CO2 and light response curves indicated that overexpression also improved photosynthetic performance relative to WT, as drought severity increased. This was not due to an effect of AOX amount on leaf water status or the development of the diffusive limitations that occur due to drought. Rather, AOX overexpression dampened photosystem stoichiometry adjustments and losses of key photosynthetic components that occurred in WT. The results indicate that AOX amount influences RL , particularly during severe drought, when cytochrome pathway respiration may become increasingly restricted. This impacts the chloroplast redox state, influencing how the photosynthetic apparatus responds to increasing drought severity. In particular, the development of biochemical limitations to photosynthesis are dampened in plants with increased nonenergy conserving RL . PMID:26032897

  7. Agroinfiltration by cytokinin-producing Agrobacterium sp. strain GV3101 primes defense responses in Nicotiana tabacum.

    PubMed

    Sheikh, Arsheed Hussain; Raghuram, Badmi; Eschen-Lippold, Lennart; Scheel, Dierk; Lee, Justin; Sinha, Alok Krishna

    2014-11-01

    Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies. PMID:25054409

  8. Role of brassinosteroid signaling in modulating Tobacco mosaic virus resistance in Nicotiana benthamiana

    PubMed Central

    Deng, Xing-Guang; Zhu, Tong; Peng, Xing-Ji; Xi, De-Hui; Guo, Hongqing; Yin, Yanhai; Zhang, Da-Wei; Lin, Hong-Hui

    2016-01-01

    Plant steroid hormones, brassinosteroids (BRs), play essential roles in plant growth, development and stress responses. However, mechanisms by which BRs interfere with plant resistance to virus remain largely unclear. In this study, we used pharmacological and genetic approaches in combination with infection experiments to investigate the role of BRs in plant defense against Tobacco Mosaic Virus (TMV) in Nicotiana benthamiana. Exogenous applied BRs enhanced plant resistance to virus infection, while application of Bikinin (inhibitor of glycogen synthase kinase-3), which activated BR signaling, increased virus susceptibility. Silencing of NbBRI1 and NbBSK1 blocked BR-induced TMV resistance, and silencing of NbBES1/BZR1 blocked Bikinin-reduced TMV resistance. Silencing of NbMEK2, NbSIPK and NbRBOHB all compromised BR-induced virus resistance and defense-associated genes expression. Furthermore, we found MEK2-SIPK cascade activated while BES1/BZR1 inhibited RBOHB-dependent ROS production, defense gene expression and virus resistance induced by BRs. Thus, our results revealed BR signaling had two opposite effects on viral defense response. On the one hand, BRs enhanced virus resistance through MEK2-SIPK cascade and RBOHB-dependent ROS burst. On the other hand, BES1/BZR1 inhibited RBOHB-dependent ROS production and acted as an important mediator of the trade-off between growth and immunity in BR signaling. PMID:26838475

  9. Population genetic structure of Myzus persicae nicotianae (Hemiptera: Aphididae) in China by microsatellite analysis.

    PubMed

    Zhao, C; Yang, X M; Tang, S H; Xu, P J; Bian, W J; Wang, X F; Wang, X W; Ren, G W

    2015-12-17

    The tobacco aphid, Myzus persicae nicotianae (Hemiptera: Aphididae), is an important agricultural pest that feeds on host plants and transmits plant viruses in China. To effectively control this pest, we investigated the genetic variation and genetic structure of 54 populations of tobacco aphids collected in China, using five microsatellite loci. An average of 7 alleles with effective number ranging from 1.5 to 6.6 was detected using these five loci, and the average polymorphic information content (PIC) was 0.652, suggesting that the five selected microsatellite loci were polymorphic and suitable for the study of population genetics. The expected heterozygosities in the populations studied ranged from 0.128 and 0.653, with an average value of 0.464. However, the observed heterozygosities ranged from 0.250 and 0.942 (average = 0.735), revealing a high genetic variability and heterozygosity excess in the Chinese tobacco aphid populations. The global fixation index (F(ST)) and mean gene flow (N(m)) were 0.34 (P < 0.0001) and 0.50, respectively, suggesting the high genetic differentiation among Chinese populations. The 54 populations of tobacco aphids were classified into two groups. The populations did not cluster geographically, as populations from the same provinces were usually present in different clusters. This was also confirmed by the Mantel test, which showed no significant correlation between the genetic distance and geographical distance or altitude. Long distance migration might be responsible for the lack of distance-related isolation.

  10. β-Carboxysomal proteins assemble into highly organized structures in Nicotiana chloroplasts.

    PubMed

    Lin, Myat T; Occhialini, Alessandro; Andralojc, P John; Devonshire, Jean; Hines, Kevin M; Parry, Martin A J; Hanson, Maureen R

    2014-07-01

    The photosynthetic efficiency of C3 plants suffers from the reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) with O2 instead of CO2 , leading to the costly process of photorespiration. Increasing the concentration of CO2 around Rubisco is a strategy used by photosynthetic prokaryotes such as cyanobacteria for more efficient incorporation of inorganic carbon. Engineering the cyanobacterial CO2 -concentrating mechanism, the carboxysome, into chloroplasts is an approach to enhance photosynthesis or to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. We have chosen to explore the possibility of producing β-carboxysomes from Synechococcus elongatus PCC7942, a model freshwater cyanobacterium. Using the agroinfiltration technique, we have transiently expressed multiple β-carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO and CcmN) in Nicotiana benthamiana with fusions that target these proteins into chloroplasts, and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the β-carboxysome are able to assemble in plant chloroplasts into highly organized assemblies resembling empty microcompartments. We demonstrate that a foreign protein can be targeted with a 17-amino-acid CcmN peptide to the shell proteins inside chloroplasts. Our experiments establish the feasibility of introducing carboxysomes into chloroplasts for the potential compartmentalization of Rubisco or other proteins.

  11. Nicotiana tabacum Tsip1-Interacting Ferredoxin 1 Affects Biotic and Abiotic Stress Resistance

    PubMed Central

    Huh, Sung Un; Lee, In-Ju; Ham, Byung-Kook; Paek, Kyung-Hee

    2012-01-01

    Tsip1, a Zn finger protein that was isolated as a direct interactor with tobacco stress-induced 1 (Tsi1), plays an important role in both biotic and abiotic stress signaling. To further understand Tsip1 function, we searched for more Tsip1-interacting proteins by yeast two-hybrid screening using a tobacco cDNA library. Screening identified a new Tsip1-interacting protein, Nicotiana tabacum Tsip1-interacting ferredoxin 1 (NtTfd1), and binding specificity was confirmed both in vitro and in vivo. The four repeats of a cysteine-rich motif (CXXCXGXG) of Tsip1 proved important for binding to NtTfd1. Virus-induced gene silencing of NtTfd1, Tsip1, and NtTfd1/Tsip1 rendered plants more susceptible to salinity stress compared with TRV2 control plants. NtTfd1- and Tsip1-silenced tobacco plants were more susceptible to infection by Cucumber mosaic virus compared with control plants. These results suggest that NtTfd1 might be involved in the regulation of biotic and abiotic stresses in chloroplasts by interaction with Tsip1. PMID:22699755

  12. Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants.

    PubMed

    Dietrich, Christof; Maiss, Edgar

    2003-10-01

    The distribution of potyviruses in mixed infected Nicotiana benthamiana plants was investigated by using green and red fluorescent proteins (GFP, DsRed). Full-length cDNA clones of Plum pox virus (PPV-NAT-AgfpS; PPV-NAT-red), Tobacco vein mottling virus (TVMV-gfp; TVMV-red) and Clover yellow vein virus (ClYVV-GFP) expressing fluorescent proteins, referred to here as labelled viruses, were used to characterize the distribution of different potyviral populations (e.g. TVMV-gfp/PPV-NAT-red), as well as populations of identical, but differently labelled potyviruses (e.g. PPV-NAT-AgfpS/PPV-NAT-red) or in mixed infections of potyviruses with labelled Potato virus X (PVX). Plants infected by any of the PVX/potyvirus combinations exhibited synergistic symptoms and large numbers of cells were doubly infected. In contrast, co-infections of differently labelled potyvirus populations appeared non-synergistic and remained predominantly separate in the infected plants, independent of whether different viruses or identical but differently labelled viruses were co-infecting. Contact of differently labelled virus populations that exhibited spatial separation was restricted to a small number of cells at the border of different fluorescent cell clusters.

  13. Isoprene emission protects photosynthesis but reduces plant productivity during drought in transgenic tobacco (Nicotiana tabacum) plants.

    PubMed

    Ryan, Annette C; Hewitt, C Nicholas; Possell, Malcolm; Vickers, Claudia E; Purnell, Anna; Mullineaux, Philip M; Davies, William J; Dodd, Ian C

    2014-01-01

    Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear. Water was withheld from transgenic isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid (ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species (ROS) and lipid peroxidation levels. Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31-46%) and lipid peroxidation (30-47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency (WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting. Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8-24% more biomass under drought, implying that isoprene emission incurred a yield penalty.

  14. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR. PMID:27143320

  15. Salivary proteins of spider mites suppress defenses in Nicotiana benthamiana and promote mite reproduction.

    PubMed

    Villarroel, Carlos A; Jonckheere, Wim; Alba, Juan M; Glas, Joris J; Dermauw, Wannes; Haring, Michel A; Van Leeuwen, Thomas; Schuurink, Robert C; Kant, Merijn R

    2016-04-01

    Spider mites (Tetranychidae sp.) are widely occurring arthropod pests on cultivated plants. Feeding by the two-spotted spider mite T. urticae, a generalist herbivore, induces a defense response in plants that mainly depends on the phytohormones jasmonic acid and salicylic acid (SA). On tomato (Solanum lycopersicum), however, certain genotypes of T. urticae and the specialist species T. evansi were found to suppress these defenses. This phenomenon occurs downstream of phytohormone accumulation via an unknown mechanism. We investigated if spider mites possess effector-like proteins in their saliva that can account for this defense suppression. First we performed an in silico prediction of the T. urticae and the T. evansi secretomes, and subsequently generated a short list of candidate effectors based on additional selection criteria such as life stage-specific expression and salivary gland expression via whole mount in situ hybridization. We picked the top five most promising protein families and then expressed representatives in Nicotiana benthamiana using Agrobacterium tumefaciens transient expression assays to assess their effect on plant defenses. Four proteins from two families suppressed defenses downstream of the phytohormone SA. Furthermore, T. urticae performance on N. benthamiana improved in response to transient expression of three of these proteins and this improvement was similar to that of mites feeding on the tomato SA accumulation mutant nahG. Our results suggest that both generalist and specialist plant-eating mite species are sensitive to SA defenses but secrete proteins via their saliva to reduce the negative effects of these defenses. PMID:26946468

  16. Nicotiana tabacum protoplasts secretome can evidence relations among regulatory elements of exocytosis mechanisms.

    PubMed

    Ul-Rehman, Reiaz; Rinalducci, Sara; Zolla, Lello; Dalessandro, Giuseppe; Di Sansebastiano, Gian Pietro

    2011-08-01

    An alternative study involving proteome analysis of the 24 hour Nicotiana tabacum protoplast culture medium was performed with the aim to confirm relations among regulatory elements of exocytotic processes. Protoplasts present many convenient features to study cellular processes during transient over-expression or suppression of specific gene's products. We performed a proteomic analysis of the culture medium fraction of protoplasts transiently expressing transgenes for 24 hours to characterize the effect of various regulatory proteins dominant negative mutants. A total number of 49 spots were found reproducible in the medium. 24 of these spots were identified with nano RP-HPLC-ESI-MS/MS. Only three and six spots were respectively identified as canonical and non-canonical secreted cell wall proteins. The low number of spots present in the culture medium fraction allowed us the ambitious experiment to analyze the influence of various SNAREs (SYP121, SYP122, SNAP33) and Rab (Rab11) dominant negative mutants. Missing a reasonable number of identified proteins the analyses gave rise to a similarity matrix statistically analyzed considering variation within the presence of 24 spots reproducible in presence of transient over-expression of SNAREs (SYP121 and SYP122) and Rab11 native cDNAs. The similarity confirmed the closer relation between the function of SYP122 and Rab11 as evidenced by the secRGUS based analysis. This analysis included the effect of SNAP33 DN mutant and showed that this Qb-c-SNARE influence both SYP121 and SYP122 SNARE complexes.

  17. Characterization of in vivo functions of Nicotiana benthamiana RabE1.

    PubMed

    Ahn, Chang Sook; Han, Jeong-A; Pai, Hyun-Sook

    2013-01-01

    We characterized the gene expression, subcellular localization, and in vivo functions of a Nicotiana benthamiana small GTPase belonging to the RabE family, designated NbRabE1. The NbRabE1 promoter drove strong β-glucuronidase reporter expression in young tissues containing actively dividing cells and in stomata guard cells. GFP fusion proteins of NbRabE1 and its dominant-negative and constitutively active mutants were all localized to the Golgi apparatus and the plasma membrane but showed different affinities for membrane attachment. Virus-induced gene silencing of NbRabE1 resulted in pleiotropic phenotypes, including growth arrest, premature senescence, and abnormal leaf development. At the cellular level, the leaves in which NbRabE1 was silenced contained abnormal stomata that lacked pores or contained incomplete ventral walls, suggesting that NbRabE1 deficiency leads to defective guard cell cytokinesis. Ectopic expression of the dominant-negative mutant of NbRabE1 in Arabidopsis thaliana resulted in retardation of shoot and root growth accompanied by defective root hair formation. These developmental defects are discussed in conjunction with proposed functions of RabE GTPases in polarized secretory vesicle trafficking.

  18. Recombinant-antibody-mediated resistance against Tomato yellow leaf curl virus in Nicotiana benthamiana.

    PubMed

    Safarnejad, Mohammad Reza; Fischer, Rainer; Commandeur, Ulrich

    2009-01-01

    Tomato yellow leaf curl virus (TYLCV) is a geminivirus species whose members cause severe crop losses in the tropics and subtropics. We report the expression of a single-chain variable fragment (scFv) antibody that protected Nicotiana benthamiana plants from a prevalent Iranian isolate of the virus (TYLCV-Ir). Two recombinant antibodies (scFv-ScRep1 and scFv-ScRep2) interacting with the multifunctional replication initiator protein (Rep) were obtained from phage display libraries and expressed in plants, both as stand-alone proteins and as N-terminal GFP fusions. Initial results indicated that both scFvs and both fusions accumulated to a detectable level in the cytosol and nucleus of plant cells. Transgenic plants challenged with TYLCV-Ir showed that the scFv-ScRep1, but more so the fusion proteins, were able to suppress TYLCV-Ir replication. These results show that expression of a scFv-ScRep1-GFP fusion protein can attenuate viral DNA replication and prevent the development of disease symptoms. The present article describes the first successful application of a recombinant antibody-mediated resistance approach against a plant DNA virus. PMID:19234665

  19. Improved photosynthetic performance during severe drought in Nicotiana tabacum overexpressing a nonenergy conserving respiratory electron sink.

    PubMed

    Dahal, Keshav; Martyn, Greg D; Vanlerberghe, Greg C

    2015-10-01

    Chloroplasts have means to manage excess reducing power but these mechanisms may become restricted by rates of ATP turnover. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that uncouples the consumption of reducing power from ATP synthesis. Physiological and biochemical analyses were used to compare respiration and photosynthesis of Nicotiana tabacum wild-type (WT) plants with that of transgenic lines overexpressing AOX, under both well-watered and drought stress conditions. With increasing drought severity, AOX overexpression acted to increase respiration in the light (RL ) relative to WT. CO2 and light response curves indicated that overexpression also improved photosynthetic performance relative to WT, as drought severity increased. This was not due to an effect of AOX amount on leaf water status or the development of the diffusive limitations that occur due to drought. Rather, AOX overexpression dampened photosystem stoichiometry adjustments and losses of key photosynthetic components that occurred in WT. The results indicate that AOX amount influences RL , particularly during severe drought, when cytochrome pathway respiration may become increasingly restricted. This impacts the chloroplast redox state, influencing how the photosynthetic apparatus responds to increasing drought severity. In particular, the development of biochemical limitations to photosynthesis are dampened in plants with increased nonenergy conserving RL .

  20. In vitro anthelmintic effect of Tobacco (Nicotiana tabacum) extract on parasitic nematode, Marshallagia marshalli.

    PubMed

    Nouri, Fatemeh; Nourollahi-Fard, Saeid R; Foroodi, Hamid R; Sharifi, Hamid

    2016-09-01

    Because of developing resistance to the existing anthelmintic drugs, there is a need for new anthelmintic agents. Tobacco plant has alkaloid materials that have antiparasitic effect. We investigated the in vitro anthelminthic effect of aqueous and alcoholic extract of Tobacco (Nicotiana tabacum) against M. marshalli. For investigating this effect, we prepared three dilutions of aqueous and alcoholic extract of Tobacco (25, 50 and 75 mg/ml). The worms exposed to extracts for 10 h at 25-30 °C. The buffer PBS used as negative control and 50 mg/ml dilution of Levamisole used as standard reference. In each group, 50 worms were examined. We used an inhibition mobility test for our study. Survival analysis with Cox proportional hazard model was used for data analysis. The result showed that compared with Levamisole 50 mg/ml, dilution of 25 and 50 mg/ml of the aqueous extract had the same anthelminthic effects (P > 0.05), but 75 mg/ml dilution of the aqueous extract and dilution of 25, 50 and 75 mg/ml of alcoholic extract had more anthelminthic effect (P < 0.05). Overall, extracts of Tobacco possess considerable anthelminthic activity and more potent effects were observed with the highest concentrations. Therefore, the in vivo study on Tobocco in animal models is recommended. PMID:27605759

  1. Reduced Susceptibility to Xanthomonas citri in Transgenic Citrus Expressing the FLS2 Receptor From Nicotiana benthamiana.

    PubMed

    Hao, Guixia; Pitino, Marco; Duan, Yongping; Stover, Ed

    2016-02-01

    Overexpression of plant pattern-recognition receptors by genetic engineering provides a novel approach to enhance plant immunity and broad-spectrum disease resistance. Citrus canker disease associated with Xanthomonas citri is one of the most important diseases damaging citrus production worldwide. In this study, we cloned the FLS2 gene from Nicotiana benthamiana cDNA and inserted it into the binary vector pBinPlus/ARS to transform Hamlin sweet orange and Carrizo citrange. Transgene presence was confirmed by polymerase chain reaction (PCR) and gene expression of NbFLS2 was compared by reverse transcription quantitative PCR. Reactive oxygen species (ROS) production in response to flg22Xcc was detected in transgenic Hamlin but not in nontransformed controls. Low or no ROS production was detected from nontransformed Hamlin seedlings challenged with flg22Xcc. Transgenic plants highly expressing NbFLS2 were selected and were evaluated for resistance to canker incited by X. citri 3213. Our results showed that the integration and expression of the NbFLS2 gene in citrus can increase canker resistance and defense-associated gene expression when challenged with X. citri. These results suggest that canker-susceptible Citrus genotypes lack strong basal defense induced by X. citri flagellin and the resistance of these genotypes can be enhanced by transgenic expression of the flagellin receptor from a resistant species.

  2. A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

    PubMed

    Zhang, Meixiang; Ahmed Rajput, Nasir; Shen, Danyu; Sun, Peng; Zeng, Wentao; Liu, Tingli; Juma Mafurah, Joseph; Dou, Daolong

    2015-06-03

    Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.

  3. An efficient Potato virus X -based microRNA silencing in Nicotiana benthamiana

    PubMed Central

    Zhao, Jinping; Liu, Qingtao; Hu, Pu; Jia, Qi; Liu, Na; Yin, Kangquan; Cheng, Ye; Yan, Fei; Chen, Jianping; Liu, Yule

    2016-01-01

    Plant microRNAs (miRNAs) play pivotal roles in many biological processes. Although many miRNAs have been identified in various plant species, the functions of these miRNAs remain largely unknown due to the shortage of effective genetic tools to block their functional activity. Recently, miRNA target mimic (TM) technologies have been applied to perturb the activity of specific endogenous miRNA or miRNA families. We previously reported that Tobacco rattle virus (TRV)-based TM expression can successfully mediate virus-based miRNA silencing/suppression (VbMS) in plants. In this study, we show the Potato virus X (PVX)-based TM expression causes strong miRNA silencing in Nicotiana benthamiana. The PVX-based expression of short tandem target mimic (STTMs) against miR165/166 and 159 caused the corresponding phenotype in all infected plants. Thus, a PVX-based VbMS is a powerful method to study miRNA function and may be useful for high-throughput investigation of miRNA function in N. benthamiana. PMID:26837708

  4. In vitro anthelmintic effect of Tobacco (Nicotiana tabacum) extract on parasitic nematode, Marshallagia marshalli.

    PubMed

    Nouri, Fatemeh; Nourollahi-Fard, Saeid R; Foroodi, Hamid R; Sharifi, Hamid

    2016-09-01

    Because of developing resistance to the existing anthelmintic drugs, there is a need for new anthelmintic agents. Tobacco plant has alkaloid materials that have antiparasitic effect. We investigated the in vitro anthelminthic effect of aqueous and alcoholic extract of Tobacco (Nicotiana tabacum) against M. marshalli. For investigating this effect, we prepared three dilutions of aqueous and alcoholic extract of Tobacco (25, 50 and 75 mg/ml). The worms exposed to extracts for 10 h at 25-30 °C. The buffer PBS used as negative control and 50 mg/ml dilution of Levamisole used as standard reference. In each group, 50 worms were examined. We used an inhibition mobility test for our study. Survival analysis with Cox proportional hazard model was used for data analysis. The result showed that compared with Levamisole 50 mg/ml, dilution of 25 and 50 mg/ml of the aqueous extract had the same anthelminthic effects (P > 0.05), but 75 mg/ml dilution of the aqueous extract and dilution of 25, 50 and 75 mg/ml of alcoholic extract had more anthelminthic effect (P < 0.05). Overall, extracts of Tobacco possess considerable anthelminthic activity and more potent effects were observed with the highest concentrations. Therefore, the in vivo study on Tobocco in animal models is recommended.

  5. Salivary proteins of spider mites suppress defenses in Nicotiana benthamiana and promote mite reproduction.

    PubMed

    Villarroel, Carlos A; Jonckheere, Wim; Alba, Juan M; Glas, Joris J; Dermauw, Wannes; Haring, Michel A; Van Leeuwen, Thomas; Schuurink, Robert C; Kant, Merijn R

    2016-04-01

    Spider mites (Tetranychidae sp.) are widely occurring arthropod pests on cultivated plants. Feeding by the two-spotted spider mite T. urticae, a generalist herbivore, induces a defense response in plants that mainly depends on the phytohormones jasmonic acid and salicylic acid (SA). On tomato (Solanum lycopersicum), however, certain genotypes of T. urticae and the specialist species T. evansi were found to suppress these defenses. This phenomenon occurs downstream of phytohormone accumulation via an unknown mechanism. We investigated if spider mites possess effector-like proteins in their saliva that can account for this defense suppression. First we performed an in silico prediction of the T. urticae and the T. evansi secretomes, and subsequently generated a short list of candidate effectors based on additional selection criteria such as life stage-specific expression and salivary gland expression via whole mount in situ hybridization. We picked the top five most promising protein families and then expressed representatives in Nicotiana benthamiana using Agrobacterium tumefaciens transient expression assays to assess their effect on plant defenses. Four proteins from two families suppressed defenses downstream of the phytohormone SA. Furthermore, T. urticae performance on N. benthamiana improved in response to transient expression of three of these proteins and this improvement was similar to that of mites feeding on the tomato SA accumulation mutant nahG. Our results suggest that both generalist and specialist plant-eating mite species are sensitive to SA defenses but secrete proteins via their saliva to reduce the negative effects of these defenses.

  6. Arabinogalactan-Proteins of the Female Sexual Tissue of Nicotiana alata

    PubMed Central

    Gell, Andrew C.; Bacic, Antony; Clarke, Adrienne E.

    1986-01-01

    Arabinogalactan-proteins (AGPs), isolated from the pistils of Nicotiana alata, an ornamental tobacco, are developmentally regulated. Both the total amount and concentration of AGP in the stigma increase during flower development, reaching 10 micrograms AGP/stigma at maturity. In contrast, AGP concentration in the style remains constant throughout the maturation period reaching 12 micrograms AGP/style at maturity. The classes of AGP present in the stigma and style during flower development, separated according to their charge by crossed-electrophoresis, are different and change during development. Pollination of flowers of N. alata with compatible or incompatible pollen results in a significant and reproducible increase in the amount of AGPs in the stigma, but not the style, compared with control unpollinated pistils. Pollination with ethanol vapor inactivated pollen also results in an increase in the amount of AGP in the stigma, but this is less than half that observed following pollination with viable pollen. There are no significant differences in the classes of AGP, based on crossed-electrophoresis, present in the pistil following pollination. Images Fig. 1 Fig. 2 PMID:16665162

  7. Transcriptome Analysis of Nicotiana tabacum Infected by Cucumber mosaic virus during Systemic Symptom Development

    PubMed Central

    Kong, Jun; Chen, Ling-Na; Qiu, Yan-Hong; Li, Gui-Fen; Meng, Xiao-Hua; Zhu, Shui-Fang

    2012-01-01

    Virus infection of plants may induce a variety of disease symptoms. However, little is known about the molecular mechanism of systemic symptom development in infected plants. Here we performed the first next-generation sequencing study to identify gene expression changes associated with disease development in tobacco plants (Nicotiana tabacum cv. Xanthi nc) induced by infection with the M strain of Cucumber mosaic virus (M-CMV). Analysis of the tobacco transcriptome by RNA-Seq identified 95,916 unigenes, 34,408 of which were new transcripts by database searches. Deep sequencing was subsequently used to compare the digital gene expression (DGE) profiles of the healthy plants with the infected plants at six sequential disease development stages, including vein clearing, mosaic, severe chlorosis, partial and complete recovery, and secondary mosaic. Thousands of differentially expressed genes were identified, and KEGG pathway analysis of these genes suggested that many biological processes, such as photosynthesis, pigment metabolism and plant-pathogen interaction, were involved in systemic symptom development. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in virus-infected plants. This information will help further our understanding of the detailed mechanisms of plant responses to viral infection. PMID:22952684

  8. Larval Helicoverpa zea Transcriptional, Growth and Behavioral Responses to Nicotine and Nicotiana tabacum

    PubMed Central

    Gog, Linus; Vogel, Heiko; Hum-Musser, Sue M.; Tuter, Jason; Musser, Richard O.

    2014-01-01

    The polyphagous feeding habits of the corn earworm, Helicoverpa zea (Boddie), underscore its status as a major agricultural pest with a wide geographic distribution and host plant repertoire. To study the transcriptomic response to toxins in diet, we conducted a microarray analysis of H. zea caterpillars feeding on artificial diet, diet laced with nicotine and Nicotiana tabacum (L.) plants. We supplemented our analysis with growth and aversion bioassays. The transcriptome reflects an abundant expression of proteases, chitin, cytochrome P450 and immune-related genes, many of which are shared between the two experimental treatments. However, the tobacco treatment tended to elicit stronger transcriptional responses than nicotine-laced diet. The salivary factor glucose oxidase, known to suppress nicotine induction in the plant, was upregulated by H. zea in response to tobacco but not to nicotine-laced diet. Reduced caterpillar growth rates accompanied the broad regulation of genes associated with growth, such as juvenile hormone epoxide hydrolase. The differential expression of chemosensory proteins, such as odorant binding-protein-2 precursor, as well as the neurotransmitter nicotinic-acetylcholine-receptor subunit 9, highlights candidate genes regulating aversive behavior towards nicotine. We suggest that an observed coincidental rise in cannibalistic behavior and regulation of proteases and protease inhibitors in H. zea larvae signify a compensatory response to induced plant defenses. PMID:26462833

  9. Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana

    PubMed Central

    Pitino, Marco; Armstrong, Cheryl M.; Cano, Liliana M.; Duan, Yongping

    2016-01-01

    Candidatus Liberibacter asiaticus “Las” is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta. PMID:27458468

  10. Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana.

    PubMed

    Pitino, Marco; Armstrong, Cheryl M; Cano, Liliana M; Duan, Yongping

    2016-01-01

    Candidatus Liberibacter asiaticus "Las" is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta. PMID:27458468

  11. The alternative respiratory pathway is involved in brassinosteroid-induced environmental stress tolerance in Nicotiana benthamiana

    PubMed Central

    Deng, Xing-Guang; Zhu, Tong; Zhang, Da-Wei; Lin, Hong-Hui

    2015-01-01

    Brassinosteroids (BRs), plant steroid hormones, play essential roles in modulating cell elongation, vascular differentiation, senescence, and stress responses. However, the mechanisms by which BRs regulate plant mitochondria and resistance to abiotic stress remain largely unclear. Mitochondrial alternative oxidase (AOX) is involved in the plant response to a variety of environmental stresses. In this report, the role of AOX in BR-induced tolerance against cold, polyethylene glycol (PEG), and high-light stresses was investigated. Exogenous applied brassinolide (BL, the most active BR) induced, while brassinazole (BRZ, a BR biosynthesis inhibitor) reduced alternative respiration and AOX1 expression in Nicotiana benthamiana. Chemical scavenging of H2O2 and virus-induced gene silencing (VIGS) of NbRBOHB compromised the BR-induced alternative respiratory pathway, and this result was further confirmed by NbAOX1 promoter analysis. Furthermore, inhibition of AOX activity by chemical treatment or a VIGS-based approach decreased plant resistance to environmental stresses and compromised BR-induced stress tolerance. Taken together, our results indicate that BR-induced AOX capability might contribute to the avoidance of superfluous reactive oxygen species accumulation and the protection of photosystems under stress conditions in N. benthamiana. PMID:26175355

  12. Transient fusion and selective secretion of vesicle proteins in Phytophthora nicotianae zoospores.

    PubMed

    Zhang, Weiwei; Blackman, Leila M; Hardham, Adrienne R

    2013-01-01

    Secretion of pathogen proteins is crucial for the establishment of disease in animals and plants. Typically, early interactions between host and pathogen trigger regulated secretion of pathogenicity factors that function in pathogen adhesion and host penetration. During the onset of plant infection by spores of the Oomycete, Phytophthora nicotianae, proteins are secreted from three types of cortical vesicles. Following induction of spore encystment, two vesicle types undergo full fusion, releasing their entire contents onto the cell surface. However, the third vesicle type, so-called large peripheral vesicles, selectively secretes a small Sushi domain-containing protein, PnCcp, while retaining a large glycoprotein, PnLpv, before moving away from the plasma membrane. Selective secretion of PnCcp is associated with its compartmentalization within the vesicle periphery. Pharmacological inhibition of dynamin function, purportedly in vesicle fission, by dynasore treatment provides evidence that selective secretion of PnCcp requires transient fusion of the large peripheral vesicles. This is the first report of selective protein secretion via transient fusion outside mammalian cells. Selective secretion is likely to be an important aspect of plant infection by this destructive pathogen. PMID:24392285

  13. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

  14. The alternative respiratory pathway is involved in brassinosteroid-induced environmental stress tolerance in Nicotiana benthamiana.

    PubMed

    Deng, Xing-Guang; Zhu, Tong; Zhang, Da-Wei; Lin, Hong-Hui

    2015-10-01

    Brassinosteroids (BRs), plant steroid hormones, play essential roles in modulating cell elongation, vascular differentiation, senescence, and stress responses. However, the mechanisms by which BRs regulate plant mitochondria and resistance to abiotic stress remain largely unclear. Mitochondrial alternative oxidase (AOX) is involved in the plant response to a variety of environmental stresses. In this report, the role of AOX in BR-induced tolerance against cold, polyethylene glycol (PEG), and high-light stresses was investigated. Exogenous applied brassinolide (BL, the most active BR) induced, while brassinazole (BRZ, a BR biosynthesis inhibitor) reduced alternative respiration and AOX1 expression in Nicotiana benthamiana. Chemical scavenging of H2O2 and virus-induced gene silencing (VIGS) of NbRBOHB compromised the BR-induced alternative respiratory pathway, and this result was further confirmed by NbAOX1 promoter analysis. Furthermore, inhibition of AOX activity by chemical treatment or a VIGS-based approach decreased plant resistance to environmental stresses and compromised BR-induced stress tolerance. Taken together, our results indicate that BR-induced AOX capability might contribute to the avoidance of superfluous reactive oxygen species accumulation and the protection of photosystems under stress conditions in N. benthamiana.

  15. Shading Influence on the Sterol Balance of Nicotiana tabacum L. 1

    PubMed Central

    Grunwald, Claus

    1978-01-01

    Tobacco plants (Nicotiana tabacum L.) were grown in the field and the apex was removed at the 42-day stage. Shading screens were set up which produced 0, 26, 67, and 90% shade. Plants were grown an additional 25 days before leaves from top, middle, and bottom stalk positions were harvested. Each leaf group was analyzed for free sterol, steryl ester, steryl glycoside, and acylsteryl glycoside. The free sterol content was lowest in top leaves and highest in bottom leaves; however, the top leaves had more steryl ester than the bottom leaves. Leaf position had no effect on steryl glycosides and acylsteryl glycosides. Shading did not influence the level of any sterol class; but in general, shading increased stigmasterol and decreased sitosterol. This trend was observed for all sterol classes, and the free sterols showed the largest and most consistent change. The younger top leaves showed a greater response than the older bottom leaves, but bottom leaves always had more stigmasterol than sitosterol even without shade. PMID:16660242

  16. Intracellular compartmentation of ions in salt adapted tobacco cells. [Nicotiana tabacum L

    SciTech Connect

    Binzel, M.L.; Hess, F.D.; Bressan, R.A.; Hasegawa, P.M. )

    1988-02-01

    Na{sup +} and Cl{sup {minus}} are the principal solutes utilized for osmotic adjustment in cells of Nicotiana tabacum L. var Wisconsin 38 (tobacco) adapted to NaCl, accumulating to levels of 472 and 386 millimolar, respectively, in cells adapted to 428 millimolar NaCl. X-ray microanalysis of unetched frozen-hydrated cells adapted to salt indicated that Na{sup +} and Cl{sup {minus}} were compartmentalized in the vacuole, at concentrations of 780 and 624 millimolar, respectively, while cytoplasmic concentrations of the ions were maintained at 96 millimolar. The morphometric differences which existed between unadapted and salt adapted cells, (cytoplasmic volume of 22 and 45% of the cell, respectively), facilitated containment of the excited volume of the x-ray signal in the cytoplasm of the adapted cells. Confirmation of ion compartmentation in salt adapted cells was obtained based on kinetic analyses of {sup 22}Na{sup +} and {sup 36}Cl{sup {minus}} efflux from cells in steady state. These data provide evidence that ion compartmentation is a component of salt adaptation of glycophyte cells.

  17. Large-scale development of PIP and SSR markers and their complementary applied in Nicotiana.

    PubMed

    Huang, L; Cao, H; Yang, L; Yu, Yu; Wang, Yu

    2013-08-01

    PIP (Potential Intron Polymorphism) and SSR (Simple Sequence Repeats) were used in many species, but large-scale development and combined use of these two markers have not been reported in tobacco. In this study, a total of 12,388 PIP and 76,848 SSR markers were designed and uploaded to a web-accessible database (http://yancao.sdau.edu.cn/tgb/). E-PCR analysis showed that PIP and SSR rarely overlapped and were strongly complementary in the tobacco genome. The density was 3.07 PIP and 1.72 SSR markers per 10 kb of the known sequences. A total of 153 and 166 alleles were detectedby 22 PIP and 22 SSR markers in 64 Nicotiana accessions. SSR produced higher PIC (polymorphism information content) values and identified more alleles than PIP, whereas PIP could identify larger numbers of rare alleles. Mantel testing demonstrated a high correlation coefficient (r = 0.949, P < 0.001) between PIP and SSR. The UPGMA dendrogram created from the combined PIP and SSR markers was clearer and more reliable than the individual PIP or SSR dendrograms. It suggested that PIP and SSR can make up the deficiency of molecular markers not only in tobacco but other plant.

  18. Proteomic profiling of cellular targets of lipopolysaccharide-induced signalling in Nicotiana tabacum BY-2 cells.

    PubMed

    Gerber, Isak B; Laukens, Kris; De Vijlder, Thomas; Witters, Erwin; Dubery, Ian A

    2008-11-01

    Plants constantly monitor for pathogen challenge and utilize a diverse array of adaptive defense mechanisms, including differential protein regulation, during pathogen attack. A proteomic analysis of Nicotiana tabacum BY-2 cells was performed in order to investigate the dynamic changes following perception of bacterial lipopolysaccharides. A multiplexed proteome analysis, employing two-dimensional difference-in-gel-electrophoresis with CyDye DIGE fluors, as well as Ruthenium II tris (bathophenanthroline disulfonate) fluorescence staining and Pro-Q Diamond phosphoprotein-specific gel staining, monitored over 1500 proteins and resulted in the identification of 88 differentially regulated proteins and phosphoproteins responsive to LPS(B.cep.)-elicitation. Functional clustering of the proteins both at the level of their abundance and phosphorylation status, revealed 9 proteins involved in transport, ion homeostasis and signal transduction. A large number of responsive proteins were found to be involved in metabolism- and energy-related processes (36), representing various metabolic pathways. Another abundant category corresponded to proteins classified as molecular chaperones and involved in protein destination/targeting (12). Other categories of proteins found to be LPS(B.cep.)-responsive and differentially regulated include cell structure- and cytoskeletal rearrangement proteins (8) and proteins involved in transcription and translation as well as degradation (11). The results indicate that LPS(B.cep.) induces metabolic reprogramming and changes in cellular activities supporting protein synthesis, -folding, vesicle trafficking and secretion; accompanied by changes to the cytoskeleton and proteosome function. Many of the identified proteins are known to be interconnected at various levels through a complex web of activation/deactivation, complex formation, protein-protein interactions, and chaperoning reactions. The presented data offers novel insights and further

  19. Effects of arbuscular mycorrhizal fungi inoculation on arsenic accumulation by tobacco (Nicotiana tabacum L.).

    PubMed

    Hua, Jianfeng; Lin, Xiangui; Yin, Rui; Jiang, Qian; Shao, Yufang

    2009-01-01

    A pot experiment was conducted to study the effects of arbuscular mycorrhizal (AM) fungi (from contaminated or uncontaminated soils) on arsenic (As) uptake of tobacco (Nicotiana tabacum L.) in As-contaminated soil. Mycorrhizal colonization rate, dry weight, As and P uptake by plants, concentrations of water-extractable As and As fractions were determined. A low mycorrhizal colonization rate (< 25%) was detected. Our research indicated that AM fungi isolated from polluted soils were no more effective than those from unpolluted soils when grown in symbiosis with tobacco. No significant differences were observed in roots and stalks dry weights among all treatments. Leaves and total plant dry weights were much higher in Glomus versiforme treatment than that in control treatment. As contents in roots and stalks from mycorrhizal treatments were much lower than that from control treatment. Total plant As content exhibited the same trend. P concentrations in tobacco were not affected by colonization, nor were stalks, leaves and total plant P contents. Roots P contents were remarkably lower in HN treatments than in other treatments. Meanwhile, decreased soil pH and lower water-extractable As concentrations and higher levels of As fraction bound to well-crystallized hydrous oxides of Fe and Al were found in mycorrhizal treatments than in controls. The protective effect of mycorrhiza against plant As uptake may be associated with changes in As solubility mediated by changing soil pH. These results indicated that under As stress, proper mechanisms employed by AM fungi can protect tobacco against As uptake. Results confirmed that AM fungi can play an important role in food quality and safety. PMID:19999968

  20. [Induction of polyploid in hairy roots of Nicotiana tabacum and its plant regeneration].

    PubMed

    Hou, Lili; Shi, Heping; Yu, Wu; Tsang, Po Keung Eric; Chow, Cheuk Fai Stephen

    2014-04-01

    By genetic transformation with Agrobacterum rhizogenes and artificial chromosome doubling techniques, we studied the induction of hairy roots and their polyploidization, and subsequent plant regeneration and nicotine determination to enhance the content of nicotine in Nicotiana tabacum. The results show that hairy roots could be induced from the basal surface of leaf explants of N. tabacum 8 days after inoculation with Agrobacterium rhizogenes ATCC15834. The percentage of the rooting leaf explants was 100% 15 days after inoculation. The hairy roots could grow rapidly and autonomously on solid or liquid phytohormones-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and paper electrophoresis of opines from N. tabacum hairy roots. The highest rate of polyploidy induction, more than 64.71%, was obtained after treatment of hairy roots with 0.1% colchicine for 36 h. The optimum medium for plant regeneration from polyploid hairy roots was MS+2.0 mg/L 6-BA +0.2 mg/L NAA. Compared with the control diploid plants, the hairy roots-regenerated plants had weak apical dominance, more axillary buds and more narrow leaves; whereas the polyploid hairy root-regenerated plants had thicker stems, shorter internodes and the colour, width and thickness of leaves were significantly higher than that of the control. Observation of the number of chromosomes in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 96 (4n = 96) chromosomes. Pot-grown experiments showed compared to the control, the flowering was delayed by 21 days in diploid hairy roots-regenerated plants and polyploid hairy root-regenerated plants. GC-MS detection shows that the content of nicotine in polyploid plants was about 6.90 and 4.57 times the control and the diploid hairy roots-regenerated plants, respectively. PMID:25195248

  1. The production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants.

    PubMed

    Limkul, Juthamard; Iizuka, Sayoko; Sato, Yohei; Misaki, Ryo; Ohashi, Takao; Ohashi, Toya; Fujiyama, Kazuhito

    2016-08-01

    For the production of therapeutic proteins in plants, the presence of β1,2-xylose and core α1,3-fucose on plants' N-glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down-regulate the endogenous N-acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco-engineered line (NbGNTI-RNAi) showed a strong reduction of plant-specific N-glycans, with the result that as much as 90.9% of the total N-glycans were of high-mannose type. Therefore, this NbGNTI-RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI-RNAi plant was cross-pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N-glycan structures that were presented on all of the four occupied N-glycosylation sites of recombinant GC in NbGNTI-RNAi plants (GC(gnt1) ) showed that the majority (ranging from 73.3% up to 85.5%) of the N-glycans had mannose-type structures lacking potential immunogenic β1,2-xylose and α1,3-fucose epitopes. Moreover, GC(gnt1) could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI-RNAi line, producing GC, was stable and the NbGNTI-RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N-glycan structures.

  2. Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis.

    PubMed

    Yuan, Ning; Yuan, Shuangrong; Li, Zhigang; Li, Dayong; Hu, Qian; Luo, Hong

    2016-01-01

    Sulfur participates in many important mechanisms and pathways of plant development. The most common source of sulfur in soil -SO4(2-)- is absorbed into root tissue and distributed into aerial part through vasculature system, where it is reduced into sulfite and finally sulfide within the subcellular organs such as chloroplasts and mitochondria and used for cysteine and methionine biosynthesis. MicroRNAs are involved in many regulation pathways by repressing the expression of their target genes. MiR395 family in Arabidopsis thaliana has been reported to be an important regulator involved in sulfate transport and assimilation, and a high-affinity sulphate transporter and three ATP sulfurylases (ATPS) were the target genes of AthmiR395 (Arabidopsis thaliana miR395). We have cloned a miR395 gene from rice (Oryza sativa) and studied its function in plant nutritional response. Our results indicated that in rice, transcript level of OsamiR395 (Oryza sativa miR395) increased under sulfate deficiency conditions, and the two predicted target genes of miR395 were down-regulated under the same conditions. Overexpression of OsamiR395h in tobacco impaired its sulfate homeostasis, and sulfate distribution was also slightly impacted among leaves of different ages. One sulfate transporter (SULTR) gene NtaSULTR2 was identified to be the target of miR395 in Nicotiana tobacum, which belongs to low affinity sulfate transporter group. Both miR395 and NtaSULTR2 respond to sulfate starvation in tobacco. PMID:27350219

  3. Profiling of Altered Metabolomic States in Nicotiana tabacum Cells Induced by Priming Agents

    PubMed Central

    Mhlongo, Msizi I.; Steenkamp, Paul A.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Dubery, Ian A.

    2016-01-01

    Metabolomics has developed into a valuable tool for advancing our understanding of plant metabolism. Plant innate immune defenses can be activated and enhanced so that, subsequent to being pre-sensitized, plants are able to launch a stronger and faster defense response upon exposure to pathogenic microorganisms, a phenomenon known as priming. Here, three contrasting chemical activators, namely acibenzolar-S-methyl, azelaic acid and riboflavin, were used to induce a primed state in Nicotiana tabacum cells. Identified biomarkers were then compared to responses induced by three phytohormones—abscisic acid, methyljasmonate, and salicylic acid. Altered metabolomes were studied using a metabolite fingerprinting approach based on liquid chromatography and mass spectrometry. Multivariate data models indicated that these inducers cause time-dependent metabolic perturbations in the cultured cells and revealed biomarkers of which the levels are affected by these agents. A total of 34 metabolites were annotated from the mass spectral data and online databases. Venn diagrams were used to identify common biomarkers as well as those unique to a specific agent. Results implicate 20 cinnamic acid derivatives conjugated to (i) quinic acid (chlorogenic acids), (ii) tyramine, (iii) polyamines, or (iv) glucose as discriminatory biomarkers of priming in tobacco cells. Functional roles for most of these metabolites in plant defense responses could thus be proposed. Metabolites induced by the activators belong to the early phenylpropanoid pathway, which indicates that different stimuli can activate similar pathways but with different metabolite fingerprints. Possible linkages to phytohormone-dependent pathways at a metabolomic level were indicated in the case of cells treated with salicylic acid and methyljasmonate. The results contribute to a better understanding of the priming phenomenon and advance our knowledge of cinnamic acid derivatives as versatile defense metabolites. PMID

  4. Glyphosate inhibition of 5-enolpyruvylshikimate 3-phosphate synthease from suspension-cultured cells of Nicotiana silvestris

    SciTech Connect

    Rubin, J.L.; Gaines, C.G.; Jensen, R.A.

    1984-07-01

    Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg, et Comes with glyphosate (N-(phosphonomethyl)glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK/sub a/ values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO/sup -/CH/sub 2/NH/sub 2//sup +/CH/sub 2/PO/sub 3//sup 2 -/, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K/sub i/ = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K/sub i/ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an (enzyme:shikimate-3-P) complex and ultimately forms the dead-end complex of (enzyme:shikimate-3-P:glyphosate). 36 references, 8 figures, 1 table.

  5. Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

    PubMed Central

    Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae Sung; Vinod, Nagarajan; Jang, Kyung Min; Kim, Sei Chang; Choi, Chang Won; Ko, Suk-Min; Jeong, Dong Kee; Udayakumar, Rajangam

    2014-01-01

    A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs. PMID:25192284

  6. Reduced gravitropic sensitivity in roots of a starch-deficient mutant of Nicotiana sylvestris

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Sack, F. D.

    1989-01-01

    Gravitropism was studied in seedlings of Nicotiana sylvestris Speg. et Comes wild-type (WT) and mutant NS 458 which has a defective plastid phosphoglucomutase (EC 2.7.5.1.). Starch was greatly reduced in NS 458 compared to the WT, but small amounts of starch were detected in rootcap columella cells in NS 458 by light and electron microscopy. The roots of WT are more sensitive to gravity than mutant NS 458 roots since: (1) in mutant roots, curvature was reduced and delayed in the time course of curvature; (2) curvature of mutant roots was 24-56% that of WT roots over the range of induction periods tested; (3) in intermittent-stimulation experiments, curvature of mutant roots was 37% or less than that of WT roots in all treatments tested. The perception time, determined by intermittent-stimulation experiments, was < or = 5 s for WT roots and 30-60 s for mutant roots. The growth rates for WT and NS 458 roots were essentially equal. These results and our previous results with WT and starchless mutant Arabidopsis roots (Kiss et al. 1989, Planta 177, 198-206) support the conclusions that a full complement of starch is necessary for full gravitropic sensitivity and that amyloplasts function in gravity perception. Since a presumed relatively small increase in plastid buoyant mass (N. sylvestris mutant versus Arabidopsis mutant) significantly improves the orientation of the N. sylvestris mutant roots, we suggest that plastids are the likeliest candidates to be triggering gravity perception in roots of both mutants.

  7. Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    PubMed Central

    Yuan, Ning; Yuan, Shuangrong; Li, Zhigang; Li, Dayong; Hu, Qian; Luo, Hong

    2016-01-01

    Sulfur participates in many important mechanisms and pathways of plant development. The most common source of sulfur in soil –SO42−– is absorbed into root tissue and distributed into aerial part through vasculature system, where it is reduced into sulfite and finally sulfide within the subcellular organs such as chloroplasts and mitochondria and used for cysteine and methionine biosynthesis. MicroRNAs are involved in many regulation pathways by repressing the expression of their target genes. MiR395 family in Arabidopsis thaliana has been reported to be an important regulator involved in sulfate transport and assimilation, and a high-affinity sulphate transporter and three ATP sulfurylases (ATPS) were the target genes of AthmiR395 (Arabidopsis thaliana miR395). We have cloned a miR395 gene from rice (Oryza sativa) and studied its function in plant nutritional response. Our results indicated that in rice, transcript level of OsamiR395 (Oryza sativa miR395) increased under sulfate deficiency conditions, and the two predicted target genes of miR395 were down-regulated under the same conditions. Overexpression of OsamiR395h in tobacco impaired its sulfate homeostasis, and sulfate distribution was also slightly impacted among leaves of different ages. One sulfate transporter (SULTR) gene NtaSULTR2 was identified to be the target of miR395 in Nicotiana tobacum, which belongs to low affinity sulfate transporter group. Both miR395 and NtaSULTR2 respond to sulfate starvation in tobacco. PMID:27350219

  8. Structural and Functional Similarities between Osmotin from Nicotiana Tabacum Seeds and Human Adiponectin

    PubMed Central

    Colonna, Giovanni

    2011-01-01

    Osmotin, a plant protein, specifically binds a seven transmembrane domain receptor-like protein to exert its biological activity via a RAS2/cAMP signaling pathway. The receptor protein is encoded in the gene ORE20/PHO36 and the mammalian homolog of PHO36 is a receptor for the human hormone adiponectin (ADIPOR1). Moreover it is known that the osmotin domain I can be overlapped to the β-barrel domain of adiponectin. Therefore, these observations and some already existing structural and biological data open a window on a possible use of the osmotin or of its derivative as adiponectin agonist. We have modelled the three-dimensional structure of the adiponectin trimer (ADIPOQ), and two ADIPOR1 and PHO36 receptors. Moreover, we have also modelled the following complexes: ADIPOQ/ADIPOR1, osmotin/PHO36 and osmotin/ADIPOR1. We have then shown the structural determinants of these interactions and their physico-chemical features and analyzed the related interaction residues involved in the formation of the complexes. The stability of the modelled structures and their complexes was always evaluated and controlled by molecular dynamics. On the basis of these results a 9 residues osmotin peptide was selected and its interaction with ADIPOR1 and PHO36 was modelled and analysed in term of energetic stability by molecular dynamics. To confirm in vivo the molecular modelling data, osmotin has been purified from nicotiana tabacum seeds and its nine residues peptide synthesized. We have used cultured human synovial fibroblasts that respond to adiponectin by increasing the expression of IL-6, TNF-alpha and IL-1beta via ADIPOR1. The biological effect on fibroblasts of osmotin and its peptide derivative has been found similar to that of adiponectin confirming the results found in silico. PMID:21311758

  9. Reconstitution of the Costunolide Biosynthetic Pathway in Yeast and Nicotiana benthamiana

    PubMed Central

    Cankar, Katarina; Goedbloed, Miriam; Charnikhova, Tatsiana; Verstappen, Francel W. A.; de Vos, Ric C. H.; Beekwilder, Jules; van der Krol, Sander; Bouwmeester, Harro J.

    2011-01-01

    The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g−1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates. PMID:21858047

  10. Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

    PubMed

    Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

    2016-01-01

    Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells. PMID:26870061

  11. Manipulation of monoubiquitin improves chilling tolerance in transgenic tobacco (Nicotiana tabacum).

    PubMed

    Feng, Yanan; Zhang, Meng; Guo, Qifang; Wang, Guokun; Gong, Jiangfeng; Xu, Ying; Wang, Wei

    2014-02-01

    Ubiquitin (Ub) is a multifunctional protein that mainly functions to tag proteins for selective degradation by the 26S proteasome. We cloned an Ub gene TaUb2 from wheat (Triticum aestivum L.) previously. To study the function of TaUB2 in chilling stress, sense and antisense Ub transgenic tobacco plants (Nicotiana tabacum L.), as well as wild type (WT) and vector control β-glucuronidase (T-GUS) plants, were used. Under stress, leaf wilting in sense plants was significantly less than in controls, but more severe in antisense plants. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) in sense plants were greater than controls, but lower in antisense plants during chilling stress and recovery. Less wilting in sense plants resulted from improved water status, which may be related to the accumulation of proline and solute sugar. Furthermore, as indicated by electrolyte leakage, membrane damage under stress was less in sense plants and more severe in antisense plants than controls. Consistent with electrolyte leakage, the malondialdehyde (MDA) content was less in sense plants, but more in antisense plants compared to controls. Meanwhile, the less accumulation of reactive oxygen species (ROS) and the greater antioxidant enzyme activity in sense plants implied the improved antioxidant competence by the overexpression of monoubiquitin gene Ta-Ub2 from wheat. We suggest that overexpressing Ub is a useful strategy to promote chilling tolerance. The improvement of ROS scavenging may be an important mechanism underlying the role of Ub in promoting plants tolerant to chilling stress.

  12. Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

    PubMed

    Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

    2016-01-01

    Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

  13. Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.

    PubMed

    Li, Jin; Stoddard, Thomas J; Demorest, Zachary L; Lavoie, Pierre-Olivier; Luo, Song; Clasen, Benjamin M; Cedrone, Frederic; Ray, Erin E; Coffman, Andrew P; Daulhac, Aurelie; Yabandith, Ann; Retterath, Adam J; Mathis, Luc; Voytas, Daniel F; D'Aoust, Marc-André; Zhang, Feng

    2016-02-01

    Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

  14. Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana.

    PubMed

    Cheng, N H; Su, C L; Carter, S A; Nelson, R S

    2000-08-01

    Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport. PMID:10929128

  15. A Multifunctional Protein Encoded by Turkey Herpesvirus Suppresses RNA Silencing in Nicotiana benthamiana▿

    PubMed Central

    Jing, Xiu-li; Fan, Mei-na; Jia, Gang; Liu, Lan-wei; Ma, Lin; Zheng, Cheng-chao; Zhu, Xiao-ping; Liu, Hong-mei; Wang, Xiao-yun

    2011-01-01

    Many plant and animal viruses counteract RNA silencing-mediated defense by encoding diverse RNA silencing suppressors. We characterized HVT063, a multifunctional protein encoded by turkey herpesvirus (HVT), as a silencing suppressor in coinfiltration assays with green fluorescent protein transgenic Nicotiana benthamiana line 16c. Our results indicated that HVT063 could strongly suppress both local and systemic RNA silencing induced by either sense RNA or double-stranded RNA (dsRNA). HVT063 could reverse local silencing, but not systemic silencing, in newly emerging leaves. The local silencing suppression activity of HVT063 was also verified using the heterologous vector PVX. Further, single alanine substitution of arginine or lysine residues of the HVT063 protein showed that each selected single amino acid contributed to the suppression activity of HVT063 and region 1 (residues 138 to 141) was more important, because three of four single amino acid mutations in this region could abolish the silencing suppressor activity of HVT063. Moreover, HVT063 seemed to induce a cell death phenotype in the infiltrated leaf region, and the HVT063 dilutions could decrease the silencing suppressor activity and alleviate the cell death phenotype. Collectively, these results suggest that HVT063 functions as a viral suppressor of RNA silencing that targets a downstream step of the dsRNA formation in the RNA silencing process. Positively charged amino acids in HVT063, such as arginine and lysine, might contribute to the suppressor activity by boosting the interaction between HVT063 and RNA, since HVT063 has been demonstrated to be an RNA binding protein. PMID:21957299

  16. Geographic variation of floral traits in Nicotiana glauca : Relationships with biotic and abiotic factors

    NASA Astrophysics Data System (ADS)

    Nattero, Julieta; Sérsic, Alicia N.; Cocucci, Andrea A.

    2011-09-01

    Geographic pattern of phenotypic variation can appear in a clinal or a mosaic fashion and can evidence adaptive or non-adaptive variation. To shed light on the mechanisms underlying this variation, we studied the relationships between geographic variation of floral traits and both biotic and abiotic factors of the hummingbird-pollinated plant, Nicotiana glauca, across its natural range. We obtained floral measures of 38 populations from an area about 1600 km long and 1050 km wide and an altitude range from 7 to over 3400 m. We used a MANOVA to detect between-population differentiations in flower traits and a DFA to determine the traits that best discriminate between populations. To test for associations between floral traits and climatic variables we used correlation analysis. We explored any possible distance-based pattern of variation (either geographic or altitudinal) in floral traits or bill length of pollinators using Mantel tests. Finally, we used a multiple regression to analyze simultaneously the effects and relative importance of abiotic predictor variables and bill length on corolla length. We found a high variation in flower traits among populations. Morphometric traits were the ones that best discriminated across populations. There was a clinal pattern of floral phenotypic variation explained by climatic factors. Differences in floral phenotypic distances were structured by altitudinal distances but not by geographic distances. Bill length of the hummingbird pollinators was structured both by altitudinal and geographic distances. Differences in bill length of hummingbird pollinators explained differences in corolla length across populations. Our findings support the assumption of flower evolution at a broad geographic scale. Floral traits seem to be structured not only by altitude but also by climatic factors.

  17. Genetic processes in intergeneric cell hybrids Atropa + Nicotiana : 1. Genetic constitution of cells of different clonal origin grown in vitro.

    PubMed

    Gleba, Y Y; Momot, V P; Okolot, A N; Cherep, N N; Skarzhynskaya, M V; Kotov, V

    1983-06-01

    The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120-150 chromosomes including 50-70 of Atropa and 50-90 of Nicotiana. Other clones are polyploid and possess 200-250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.

  18. Virus-induced gene silencing reveals signal transduction components required for the Pvr9-mediated hypersensitive response in Nicotiana benthamiana.

    PubMed

    Tran, Phu-Tri; Choi, Hoseong; Choi, Doil; Kim, Kook-Hyung

    2016-08-01

    Resistance to pathogens mediated by plant resistance (R) proteins requires different signaling transduction components and pathways. Our previous studies revealed that a potyvirus resistance gene in pepper, Pvr9, confers a hypersensitive response (HR) to pepper mottle virus in Nicotiana benthamiana. Our results show that the Pvr9-mediated HR against pepper mottle virus infection requires HSP90, SGT1, NDR1, but not EDS1. These results suggest that the Pvr9-mediated HR is possibly related to the SA pathway but not the ET, JA, ROS or NO pathways.

  19. Flower-bud formation in explants of photoperiodic and day-neutral Nicotiana biotypes and its bearing on the regulation of flower formation

    SciTech Connect

    Rajeevan, M.S.; Lang, A. )

    1993-05-15

    The capacity to form flower buds in thin-layer explants was studied in Nicotiana of several species, cultivars, and lines of differing in their response to photoperiod. This capacity was found in all biotypes examined and could extend into sepals and corolla. It varied depending on genotype, source tissue and its developmental state, and composition of the culture medium, particularly the levels of glucose, auxin, and cytokinin. It was greatest in the two day-neutral plants examined, Samsun tobacco and Nicotiana rustica, where it extended from the inflorescence region down the vegetative stem, in a basipetally decreasing gradient; it was least in the two qualitative photoperiodic plants studied, the long-day plant Nicotiana silvestris and the short-day plant Maryland Mammoth tobacco, the quantitative long-day plant Nicotiana alata and the quantitative short-day plant Nicotiana otophora line 38-G-81, where it was limited to the pedicels (and, in some cases, the sepals). Regardless of the photoperiodic response of the source plants, the response was the same in explants cultured under long and short days. The capacity to form flow buds in explants is present in all Nicotiana biotypes studied supports the idea that it is regulated by the same mechanism(s), regardless of the plant's photoperiodic character. However, flower formation in the explants is not identical with de novo flower formation in a hitherto vegetative plant: it is rather the expression of a floral state already established in the plant, although it can vary widely in extent and spatial distribution. Culture conditions that permit flower-bud formation in an explant are conditions that maintain the floral state and encourage its expression; conditions under which no flower buds are formed reduce this state and/or prevent its expression. 14 refs., 5 figs., 3 tabs.

  20. Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum

    PubMed Central

    Kohl, Thomas O; Hitzeroth, Inga I; Christensen, Neil D; Rybicki, Edward P

    2007-01-01

    Background We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine. Results Transformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and – to a lesser degree – the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 μg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 μg/g from N. tabacum plants – a significant increase over previous efforts. Immunization of New Zealand white rabbits with ~50 μg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro. Conclusion We expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11

  1. Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana

    PubMed Central

    Marín Viegas, Vanesa S.; Acevedo, Gonzalo R.; Bayardo, Mariela P.; Chirdo, Fernando G.; Petruccelli, Silvana

    2015-01-01

    Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to

  2. Ionomic profiling of Nicotiana langsdorffii wild-type and mutant genotypes exposed to abiotic stresses.

    PubMed

    Ardini, Francisco; Soggia, Francesco; Abelmoschi, Maria Luisa; Magi, Emanuele; Grotti, Marco

    2013-01-01

    To provide a new insight into the response of plants to abiotic stresses, the ionomic profiles of Nicotiana langsdorffii specimens have been determined before and after exposure to toxic metals (chromium) or drought conditions. The plants were genetically transformed with the rat glucocorticoid receptor (GR) or the gene for Agrobacterium rhizogenes rolC, because these modifications are known to produce an imbalance in phytohormone equilibria and a significant change in the defence response of the plant. Elemental profiles were obtained by developing and applying analytical procedures based on inductively coupled plasma atomic emission and mass spectrometry (ICP-AES/MS). In particular, the removal of isobaric interferences affecting the determination of Cr and V by ICP-MS was accomplished by use of a dynamic reaction cell, after optimization of the relevant conditions. The combined use of ICP atomic emission and mass spectrometry enabled the determination of 29 major and trace elements (Ba, Bi, Ca, Cd, Co, Cr, Cu, Eu, Fe, Ga, K, Li, Mg, Mn, Mo, Na, P, Pb, Pt, Rb, S, Sb, Sn, Sr, Te, V, W, Y, and Zn) in different parts of the plants (roots, stems, and leaves), with high accuracy and precision. Multivariate data processing and study of element distribution patterns provided new information about the ionomic response of the target organism to chemical treatment or water stress. Genetic modification mainly affected the distribution of Bi, Cr, Mo, Na, and S, indicating that these elements were involved in biochemical processes controlled by the GR or rolC genes. Chemical stress strongly affected accumulation of several elements (Ba, Ca, Fe, Ga, K, Li, Mn, Mo, Na, P, Pb, Rb, S, Sn, Te, V, and Zn) in different ways; for Ca, Fe, K, Mn, Na, and P the effect was quite similar to that observed in other studies after treatment with other transition elements, for example Cu and Cd. The effect of water deficit was less evident, mainly consisting in a decrease of Ba, Cr, Na, and Sr

  3. [Antioxidative response of Phytolacca americana and Nicotiana tabacum to manganese stresses].

    PubMed

    Zhang, Yu-xiu; Huang, Zhi-bo; Zhang, Hong-mei; Li, Lin-feng; Chai, Tuan-yao

    2009-12-01

    Plant species capable of accumulating heavy metals are of considerable interest for phytoremediation and phytomining. The mechanism of Mn tolerance/hyperaccumulate in Phytolacca americana L. is less known. To elucidate the role of antioxidative enzyme in response to Mn, the 6-week-old seedling of Mn hyperaccumulator P. americana and non-accumulator-tobacco (Nicotiana tabacum) were exposed to half strength Hoagland solution with 1 mmol x L(-1) or 3 mmol x L(-1) MnCl2 for 4 days. The photosynthetic rate in P. americana decreased more slowly than that in tobacco, while the MDA content and electrolyte leakage in tobacco increased more rapidly than that in P. americana. For example, after exposure to 1 mmol x L(-1) Mn for 4 days, the photosynthetic rates of P. americana and tobacco in comparison to the control reduced by 13.3% and 75.5%, respectively. The MDA content and electrolyte leakage in tobacco increased by 347.3% and 120.1%, respectively, whereas Mn had no marked effect on both of it in P. americana, indicated that the oxidative damage in tobacco was more serious than that in P. americana. The activities of SOD and POD of both species increased rapidly with elevated Mn concentration and exposure time in both species, the increase of SOD activity in P. americana was higher than that in tobacco. CAT activity in tobacco declined rapidly, while the activity of CAT in P. americana was increased. The activities of SOD, POD and CAT in P. americana upon 1 mmol x L(-1) Mn exposure increased by 161.1%, 111.3% and 17.5%, respectively. The activities of SOD and POD in tobacco increased by 55.5% and 206.0%, respectively, while CAT activity decreased by 15.6%, indicating that the antioxidative enzymes in P. americana, particularly in CAT,could fully scavenge the reactive oxygen species generated by Mn toxicity. These results collectively indicate that the enzymatic antioxidation capacity is one of the important mechanisms responsible for Mn tolerance in hyperaccumulator plant

  4. Genome-wide identification of the expansin gene family in tobacco (Nicotiana tabacum).

    PubMed

    Ding, Anming; Marowa, Prince; Kong, Yingzhen

    2016-10-01

    Expansins are pH-dependent cell wall loosening proteins which form a large family in plants. They have been shown to be involved in various developmental processes and been implicated in enabling plants' ability to absorb nutrients from the soil as well as conferring biotic and abiotic stress resistances. It is therefore clear that they can be potential targets in genetic engineering for crop improvement. Tobacco (Nicotiana tabacum) is a major crop species as well as a model organism. Considering that only a few tobacco expansins have been studied, a genome-wide analysis of the tobacco expansin gene family is necessary. In this study, we identified 52 expansins in tobacco, which were classified into four subfamilies: 36 NtEXPAs, 6 NtEXPBs, 3 NtEXLAs and 7 NtEXLBs. Compared to other species, the NtEXLB subfamily size was relatively larger. Phylogenetic analysis showed that the 52 tobacco expansins were divided into 13 subgroups. Gene structure analysis revealed that genes within subfamilies/subgroups exhibited similar characteristics such as gene structure and protein motif arrangement. Whole-genome duplication and tandem duplication events may have played important roles in the expanding of tobacco expansins. Cis-Acting element analysis revealed that each expansin gene was regulated or several expansin genes were co-regulated by both internal and environmental factors. 35 of these genes were identified as being expressed according to a microarray analysis. In contrast to most NtEXPAs which had higher expression levels in young organs, NtEXLAs and NtEXLBs were preferentially expressed in mature or senescent tissues, suggesting that they might play different roles in different organs or at different developmental stages. As the first step towards genome-wide analysis of the tobacco expansin gene family, our work provides solid background information related to structure, evolution and expression as well as regulatory cis-acting elements of the tobacco expansins. This

  5. Expression of synthetic human tropoelastin (hTE) protein in Nicotiana tabacum

    PubMed Central

    Abdelghani, Mona; El-Heba, Ghada A Abu; Abdelhadi, Abdelhadi A; Abdallah, Naglaa A

    2015-01-01

    ABSTRACT Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal.  Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium- mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5th day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3–10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at ∼70 kDa from freshly extracted protein, while two degraded bands of ∼55 and ∼45 kDa, representing a pattern of tropoelastin were appeared with frozen samples

  6. Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana.

    PubMed

    Marín Viegas, Vanesa S; Acevedo, Gonzalo R; Bayardo, Mariela P; Chirdo, Fernando G; Petruccelli, Silvana

    2015-01-01

    Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to

  7. Genome-wide identification of the expansin gene family in tobacco (Nicotiana tabacum).

    PubMed

    Ding, Anming; Marowa, Prince; Kong, Yingzhen

    2016-10-01

    Expansins are pH-dependent cell wall loosening proteins which form a large family in plants. They have been shown to be involved in various developmental processes and been implicated in enabling plants' ability to absorb nutrients from the soil as well as conferring biotic and abiotic stress resistances. It is therefore clear that they can be potential targets in genetic engineering for crop improvement. Tobacco (Nicotiana tabacum) is a major crop species as well as a model organism. Considering that only a few tobacco expansins have been studied, a genome-wide analysis of the tobacco expansin gene family is necessary. In this study, we identified 52 expansins in tobacco, which were classified into four subfamilies: 36 NtEXPAs, 6 NtEXPBs, 3 NtEXLAs and 7 NtEXLBs. Compared to other species, the NtEXLB subfamily size was relatively larger. Phylogenetic analysis showed that the 52 tobacco expansins were divided into 13 subgroups. Gene structure analysis revealed that genes within subfamilies/subgroups exhibited similar characteristics such as gene structure and protein motif arrangement. Whole-genome duplication and tandem duplication events may have played important roles in the expanding of tobacco expansins. Cis-Acting element analysis revealed that each expansin gene was regulated or several expansin genes were co-regulated by both internal and environmental factors. 35 of these genes were identified as being expressed according to a microarray analysis. In contrast to most NtEXPAs which had higher expression levels in young organs, NtEXLAs and NtEXLBs were preferentially expressed in mature or senescent tissues, suggesting that they might play different roles in different organs or at different developmental stages. As the first step towards genome-wide analysis of the tobacco expansin gene family, our work provides solid background information related to structure, evolution and expression as well as regulatory cis-acting elements of the tobacco expansins. This

  8. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance.

    PubMed

    Shi, Yanmei; Guo, Jinggong; Zhang, Wei; Jin, Lifeng; Liu, Pingping; Chen, Xia; Li, Feng; Wei, Pan; Li, Zefeng; Li, Wenzheng; Wei, Chunyang; Zheng, Qingxia; Chen, Qiansi; Zhang, Jianfeng; Lin, Fucheng; Qu, Lingbo; Snyder, John Hugh; Wang, Ran

    2015-01-01

    Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum. PMID:26703579

  9. The Cotton WRKY Gene GhWRKY41 Positively Regulates Salt and Drought Stress Tolerance in Transgenic Nicotiana benthamiana

    PubMed Central

    Chen, Xiaobo; Lu, Wenjing; Li, Han; Wang, Xiuling; Hao, Lili; Guo, Xingqi

    2015-01-01

    WRKY transcription factors constitute a very large family of proteins in plants and participate in modulating plant biological processes, such as growth, development and stress responses. However, the exact roles of WRKY proteins are unclear, particularly in non-model plants. In this study, Gossypium hirsutum WRKY41 (GhWRKY41) was isolated and transformed into Nicotiana benthamiana. Our results showed that overexpression of GhWRKY41 enhanced the drought and salt stress tolerance of transgenic Nicotiana benthamiana. The transgenic plants exhibited lower malondialdehyde content and higher antioxidant enzyme activity, and the expression of antioxidant genes was upregulated in transgenic plants exposed to osmotic stress. A β-glucuronidase (GUS) staining assay showed that GhWRKY41 was highly expressed in the stomata when plants were exposed to osmotic stress, and plants overexpressing GhWRKY41 exhibited enhanced stomatal closure when they were exposed to osmotic stress. Taken together, our findings demonstrate that GhWRKY41 may enhance plant tolerance to stress by functioning as a positive regulator of stoma closure and by regulating reactive oxygen species (ROS) scavenging and the expression of antioxidant genes. PMID:26562293

  10. Evolution of rDNA in Nicotiana Allopolyploids: A Potential Link between rDNA Homogenization and Epigenetics

    PubMed Central

    Kovarik, Ales; Dadejova, Martina; Lim, Yoong K.; Chase, Mark W.; Clarkson, James J.; Knapp, Sandra; Leitch, Andrew R.

    2008-01-01

    Background The evolution and biology of rDNA have interested biologists for many years, in part, because of two intriguing processes: (1) nucleolar dominance and (2) sequence homogenization. We review patterns of evolution in rDNA in the angiosperm genus Nicotiana to determine consequences of allopolyploidy on these processes. Scope Allopolyploid species of Nicotiana are ideal for studying rDNA evolution because phylogenetic reconstruction of DNA sequences has revealed patterns of species divergence and their parents. From these studies we also know that polyploids formed over widely different timeframes (thousands to millions of years), enabling comparative and temporal studies of rDNA structure, activity and chromosomal distribution. In addition studies on synthetic polyploids enable the consequences of de novo polyploidy on rDNA activity to be determined. Conclusions We propose that rDNA epigenetic expression patterns established even in F1 hybrids have a material influence on the likely patterns of divergence of rDNA. It is the active rDNA units that are vulnerable to homogenization, which probably acts to reduce mutational load across the active array. Those rDNA units that are epigenetically silenced may be less vulnerable to sequence homogenization. Selection cannot act on these silenced genes, and they are likely to accumulate mutations and eventually be eliminated from the genome. It is likely that whole silenced arrays will be deleted in polyploids of 1 million years of age and older. PMID:18310159

  11. A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

    PubMed

    Moghbel, Nahid; Ryu, BoMi; Steadman, Kathryn J

    2015-08-01

    A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD <2%. Limit of detection was 1.6μg/mL for nicotine and below 1μg/mL for all other alkaloids. The limit of quantification was 2.8 and 4.8μg/mL for nornicotine and nicotine respectively, and below 2μg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.

  12. Reversal by Calcium Ions of the Growth Inhibition of Debaryomyces nicotianae Caused by Antifungal Polyene Antibiotics1

    PubMed Central

    Berdicevsky, Israela; Grossowicz, Nathan

    1972-01-01

    Only Debaryomyces nicotianae strain 77, of seven different yeast strains tested, was found to be resistant to heptamycin and other antifungal heptaenes when grown in a rich medium. This strain, however, like the other six, was completely susceptible to these antibiotics in a minimal medium. Addition of yeast extract to the minimal medium abolished the heptamycin effect; calcium ions fully duplicated the effect of yeast extract; Mg2+ and Mn2+ were also effective but less so than Ca2+. Ca2+ also counteracted the activity of the heptaenes ascosin and trichomycin. Complete reversal of the polyene inhibition by Ca2+ was obtained if the cation was added simultaneously with the antibiotic; addition of Ca2+ 2 hr after the polyene was without effect. Addition of Ca2+ in the absence of the polyene caused a slight, if any, growth stimulation of D. nicotianae 77. Cholesterol also counteracted polyene activity; this was due to the formation of a complex with the antibiotic which prevented the polyene from reaching the site of action—the cytoplasmic membrane. No evidence for complex formation between heptamycin and calcium was found. The importance of Ca2+ in membrane structure, as evidenced from heptaene studies, is discussed. PMID:4598328

  13. Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum.

    PubMed

    Morita, Masahiko; Shitan, Nobukazu; Sawada, Keisuke; Van Montagu, Marc C E; Inzé, Dirk; Rischer, Heiko; Goossens, Alain; Oksman-Caldentey, Kirsi-Marja; Moriyama, Yoshinori; Yazaki, Kazufumi

    2009-02-17

    Alkaloids play a key role in plant defense mechanisms against pathogens and herbivores, but the plants themselves need to cope with their toxicity as well. The major alkaloid of the Nicotiana species, nicotine, is translocated via xylem transport from the root tissues where it is biosynthesized to the accumulation sites, the vacuoles of leaves. To unravel the molecular mechanisms behind this membrane transport, we characterized one transporter, the tobacco (Nicotiana tabacum) jasmonate-inducible alkaloid transporter 1 (Nt-JAT1), whose expression was coregulated with that of nicotine biosynthetic genes in methyl jasmonate-treated tobacco cells. Nt-JAT1, belonging to the family of multidrug and toxic compound extrusion transporters, was expressed in roots, stems, and leaves, and localized in the tonoplast of leaf cells. When produced in yeast cells, Nt-JAT1 occurred mainly in the plasma membrane and showed nicotine efflux activity. Biochemical analysis with proteoliposomes reconstituted with purified Nt-JAT1 and bacterial F(0)F(1)-ATPase revealed that Nt-JAT1 functioned as a proton antiporter and recognized endogenous tobacco alkaloids, such as nicotine and anabasine, and other alkaloids, such as hyoscyamine and berberine, but not flavonoids. These findings strongly suggest that Nt-JAT1 plays an important role in the nicotine translocation by acting as a secondary transporter responsible for unloading of alkaloids in the aerial parts and deposition in the vacuoles. PMID:19168636

  14. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

    PubMed Central

    Shi, Yanmei; Guo, Jinggong; Zhang, Wei; Jin, Lifeng; Liu, Pingping; Chen, Xia; Li, Feng; Wei, Pan; Li, Zefeng; Li, Wenzheng; Wei, Chunyang; Zheng, Qingxia; Chen, Qiansi; Zhang, Jianfeng; Lin, Fucheng; Qu, Lingbo; Snyder, John Hugh; Wang, Ran

    2015-01-01

    Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum. PMID:26703579

  15. In vitro and In vivo anthelmintic activity of Nicotiana tabacum L. leaves against gastrointestinal nematodes of sheep.

    PubMed

    Iqbal, Zafar; Lateef, Muhammad; Jabbar, Abdul; Ghayur, Muhammad Nabeel; Gilani, Anwarul Hassan

    2006-01-01

    The in vitro and in vivo anthelmintic activity of Nicotiana tabacum L. leaves was studied to rationalize its traditional use. Live Haemonchus contortus were used to assess the in vitro anthelmintic effect of a crude aqueous extract (CAE) and a methanol extract (CME) of N. tabacum. The in vitro inhibitory effect of both the extracts was evident from the paralysis and/or mortality of worms noted at 6 h post-exposure. For the in vivo studies, CAE and CME were administered in increasing doses (1.0-3.0 g/kg) to sheep naturally infected with mixed species of gastrointestinal nematodes. A maximum reduction of 73.6% in eggs per gram (EPG) of faeces was recorded on day 5 post-treatment with CME (3.0 g/kg) while the same dose of CAE showed a 49.4% reduction. Levamisole (7.5 mg/kg), a standard anthelmintic agent, showed a 99.6% reduction in EPG. These data show that the aqueous and methanol extracts of Nicotiana tabacum exhibit dose-dependent anthelmintic activity both in vitro and in vivo, thus justifying its use in the traditional medicine system of Pakistan.

  16. Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny.

    PubMed

    Thyssen, Gregory; Svab, Zora; Maliga, Pal

    2012-10-01

    Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.

  17. [Preferential localization of cadmium on the iterative DNA sequences from cultured tissues of the crown gall of tobacco (Nicotiana tabacum, var. Wisconsis 38)].

    PubMed

    Sissoëff, I; Grisvard, J; Guillé, E; Laterjet, R

    1975-05-26

    Fractionation of total crown-gall tissue culture DNA from Nicotiana tabacum by Ag+-Cs2SO4 density gradient is described. Cadmium ions determination is performed in each fraction by anodic stripping voltammetry. The cadmium content of the DNA in the lightest density fractions is 100 to 1000 times higher than in the other fractions.

  18. [Transgenic Expression of Serratia marcescens Native and Mutant Nucleases Modulates Tobacco Mosaic Virus Resistance in Nicotiana tabacum L].

    PubMed

    Trifonova, E A; Saveleva, A V; Romanova, A V; Filipenko, E A; Sapotsky, M V; Malinovsky, V I; Kochetov, A V; Shumny, V K

    2015-07-01

    Extracellular Serratia marcescens nuclease is an extremely active enzyme which non-specifically degrades RNA and DNA. Its antiviral activity was previously shown both in animals and in plants when applied exogenously. Transgenic tobacco plants (Nicotiana tabacum L cv. SR1) expressing S. marcescens chimeric, mutant, and intracellular mutant nuclease gene variants were regenerated and challenged with tobacco mosaic virus. The transgenic plants exhibited a higher level of resistance to the virus infection than the control non-transgenic plants. The resistance was evidenced by the delay of the appearance of mosaic symptoms and the retarded accumulation of viral antigen. Thus, these results reveal that modulations of both extracellular nuclease activity and intracellular RNA/DNA binding can protect plants against viral diseases. PMID:26410939

  19. Soft material-based microculture system having air permeable cover sheet for the protoplast culture of Nicotiana tabacum.

    PubMed

    Ju, Jong Il; Ko, Jung-Moon; Kim, So Hyeon; Baek, Ju Yeoul; Cha, Hyeon-Cheol; Lee, Sang Hoon

    2006-08-01

    In plant cell culture, the delivery of nutrition and gas (mainly oxygen) to the cells is the most important factor for viability. In this paper, we propose a polydimethylsiloxane (PDMS)-based microculture system that is designed to have good aeration. PDMS is known to have excellent air permeability, and through the experimental method, we investigated the relation between the degree of air delivery and the thickness of the PDMS sheet covering the culture chamber. We determined the proper thickness of the cover sheet, and cultured protoplasts of Nicotiana tabacum in a culture chamber covered with a PDMS sheet having thickness of 400 microm. The cells were successfully divided, and lived well inside the culture chamber for 10 days. In addition, protoplasts were cultured inside the culture chambers covered with the cover glass and the PDMS sheet, respectively, and the microcolonies were formed well inside the PDMS covered chamber after 10 days.

  20. Macrocyclic Trichothecenes from Myrothecium roridum Strain M10 with Motility Inhibitory and Zoosporicidal Activities against Phytophthora nicotianae.

    PubMed

    Mondol, Muhammad Abdul Mojid; Surovy, Musrat Zahan; Islam, M Tofazzal; Schüffler, Anja; Laatsch, Hartmut

    2015-10-14

    The cytotoxicity of the extract obtained from Myrothecium roridum M10 and a characteristic (1)H signal at δH ∼8 led to the assumption that verrucarin/roridin-type compounds were present. Upscaling on rice medium led to the isolation of four new metabolites: verrucarins Y (1) and Z (6) (macrocyclic trichothecenes), bilain D (12) (a diketopiperazine derivative), and hamavellone C (14) (an unusual cyclopropyl diketone). In addition, nine known trichothecenes [verrucarin A (3), 16-hydroxyverrucarin A (5), verrucarin B (7), 16-hydroxyverrucarin B (8), verrucarin J (2), verrucarin X (4), roridin A (9), roridin L-2 (10), and trichoverritone (11)] and a bicyclic lactone [myrotheciumone A (15)] were identified. Their structures and configurations were determined by spectroscopic methods, published data, Mosher's method, and considering biosyntheses. Some trichothecenes showed motility inhibition followed by lysis of the zoospores against devastating Phytophthora nicotianae within 5 min. Compounds 2, 3, 7, and 9 also exhibited potent activities against Candida albicans and Mucor miehei.

  1. Comparison of the hypersensitive response induced by the tomato Cf-4 and Cf-9 genes in Nicotiana spp.

    PubMed

    Thomas, C M; Tang, S; Hammond-Kosack, K; Jones, J D

    2000-04-01

    We have previously shown that tomato Cf-9 induces an Avr9-dependent hypersensitive response (HR) in Nicotiana tabacum and potato. We show here that Cf-4 also induces an Avr4-dependent HR in two tobacco species (N. tabacum and N. benthamiana). The HR induced by Cf-4 and Cf-9 was compared in stable tobacco transgenics by a seedling lethal assay and resistance to recombinant Potato virus X expressing Avr4 or Avr9. We also compared HR induction with Agrobacterium-mediated transient expression. The Cf-4/Avr4 combination induced a more rapid HR than Cf-9/Avr9. Sensitive assays for Cf-9 and Cf-4 function should prove useful for structure/function analyses of these resistance proteins in tobacco. PMID:10755310

  2. Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.

    PubMed

    Hamill, J D; Robins, R J; Parr, A J; Evans, D M; Furze, J M; Rhodes, M J

    1990-07-01

    Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation. PMID:2103440

  3. Ionome changes in Xylella fastidiosa-infected Nicotiana tabacum correlate with virulence and discriminate between subspecies of bacterial isolates.

    PubMed

    Oliver, J E; Sefick, S A; Parker, J K; Arnold, T; Cobine, P A; De La Fuente, L

    2014-10-01

    Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection. PMID:24983508

  4. Genome-wide identification and expression analysis of the WRKY gene family in common tobacco (Nicotiana tabacum L.).

    PubMed

    Xiaohua, Xiang; Xinru, Wu; Jiangtao, Chao; Minglei, Yang; Fan, Yang; Guo, Chen; Guanshan, Liu; Yuanying, Wang

    2016-09-01

    The coding products of WRKY gene family plays important roles in plant growth and development as well as in various stress responses. They have been identified in various plants, but only few in common tobacco (Nicotiana tabacum L.). In this study, 164 putative WRKY proteins in the common tobacco genome were identified by using the conserved WRKY sequence (PF03106) from the Pfam database. Phylogenetic trees, functional domain analysis, chromosomal localization, subcellular localization and tissue expression patterns were analyzed with the bioinformatics softwares, including DNAMAN 5.0, Weblogo 3, MEGA 5.1, MG2C and MEME. First of all, phylogenetic trees divided all the candidate genes into three subfamilies: Ⅰ, Ⅱ and Ⅲ, respectively, and subfamily Ⅱ could be further divided into five subgroups: group Ⅱ-a, -b, -c, -d and -e. Secondly, the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK followed by a zinc-finger motif. Most of the NtWRKY genes contained 2-5 exons and a highly conserved gene structure. Thirdly, 154 out of 164 NtWRKY genes were distributed with different densities on 24 chromosomes, and each subfamily with different patterns and frequency. The largest number of NtWRKY genes was found on chromosome VI, and only one on chromosome X. Fourthly, the majority of NtWRKY members located in the nucleus, with 74 percent of subfamily Ⅲ in the extracellular matrix. Lastly, the members in the same subfamily had different spatial and temporal expression profiles, with 11 NtWRKY genes in roots, stems and leaves expressed at various levels. The expression of genes NtWRKY26, NtWRKY30 and NtWRKY32 can be induced by Phytophthora nicotianae. Our research thus provides valuable information for NtWRKY gene cloning and functional characterization in common tobacco. PMID:27644745

  5. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D

    2016-05-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana. PMID

  6. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed Central

    Dalton, Heidi L.; Blomstedt, Cecilia K.; Neale, Alan D.; Gleadow, Ros; DeBoer, Kathleen D.; Hamill, John D.

    2016-01-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana. PMID

  7. Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds.

    PubMed

    Murfett, J; McClure, B A

    1998-06-01

    Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.

  8. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D

    2016-05-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

  9. Cloning, expression analysis and recombinant expression of a gene encoding a polygalacturonase-inhibiting protein from tobacco, Nicotiana tabacum.

    PubMed

    Zhang, Chengsheng; Feng, Chao; Wang, Jing; Kong, Fanyu; Sun, Wenxiu; Wang, Fenglong

    2016-05-01

    Polygalacturonase inhibiting proteins (PGIPs) are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG) activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum), hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5'-untranslated region (UTR), and 209 bp 3'-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP's deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA), abscisic acid (ABA), salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3). Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding. PMID:27441281

  10. Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

    PubMed

    Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

    2014-01-01

    Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

  11. Differential Responses to Virus Challenge of Laboratory and Wild Accessions of Australian Species of Nicotiana, and Comparative Analysis of RDR1 Gene Sequences

    PubMed Central

    Zhang, Chao; Long, Vicki; Roossinck, Marilyn J.; Koh, Shu Hui; Jones, Michael G. K.; Iqbal, Sadia

    2015-01-01

    Nicotiana benthamiana is a model plant utilised internationally in plant virology because of its apparent hyper-susceptibility to virus infection. Previously, others showed that all laboratory accessions of N. benthamiana have a very narrow genetic basis, probably originating from a single source. It is unknown if responses to virus infection exhibited by the laboratory accession are typical of the species as a whole. To test this, 23 accessions of N. benthamiana were collected from wild populations and challenged with one to four viruses. Additionally, accessions of 21 other Nicotiana species and subspecies from Australia, one from Peru and one from Namibia were tested for susceptibility to the viruses, and for the presence of a mutated RNA-dependent RNA polymerase I allele (Nb-RDR1m) described previously from a laboratory accession of N. benthamiana. All Australian Nicotiana accessions tested were susceptible to virus infections, although there was symptom variability within and between species. The most striking difference was that plants of a laboratory accession of N. benthamiana (RA-4) exhibited hypersensitivity to Yellow tailflower mild mottle tobamovirus infection and died, whereas plants of wild N. benthamiana accessions responded with non-necrotic symptoms. Plants of certain N. occidentalis accessions also exhibited initial hypersensitivity to Yellow tailflower mild mottle virus resembling that of N. benthamiana RA-4 plants, but later recovered. The mutant Nb-RDR1m allele was identified from N. benthamiana RA-4 but not from any of 51 other Nicotiana accessions, including wild accessions of N. benthamiana, demonstrating that the accession of N. benthamiana used widely in laboratories is unusual. PMID:25822508

  12. Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

    PubMed

    Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

    2014-01-01

    Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses. PMID:25153631

  13. Ozone, sulfur dioxide, and ultraviolet B have similar effects on mRNA accumulation of antioxidant genes in Nicotiana plumbaginifolia L.

    SciTech Connect

    Willekens, H.; Van Camp, W.; Van Montagu, M.; Inze, D.; Langebartels, C.; Sandermann, H. Jr. |

    1994-11-01

    We have studied the expression of antioxidant genes in response to near ambient conditions of O{sub 3}, SO{sub 2}, and ultraviolet B (UV-B) in Nicotiana plumbaginifolia L. The genes analyzed encode four different superoxide dismutases (SODs), three catalases (Cat1, Cat2, and Cat3), the cytosolic ascorbate peroxidase (cyt APx), and glutathione peroxidase (GPx). The experimental setup for each treatment was essentially the same and caused no visible damage, thus allowing direct comparison of the different stress responses. Our data showed that the effects of O{sub 3}, SO{sub 2}, and UV-B on the antioxidant genes are very similar, although the response to SO{sub 2} is generally less pronounced and delayed. The effects of the different stresses are characterized by a decline in Cat1, a moderate increase in Cat3, and a strong increase in Cat2 and GPx. Remarkably, SODs and cyt APx were not affected. Analysis of SOD and APx expression in the ozone-sensitive Nicotiana tabacum L. cv PBD6 revealed that induction of the cytosolic copper/zinc SOD and cyt APx occurs only with the onset of visible damage. It is proposed that alterations in mRNA levels of catalases and GPx, but not of SODs and cyt APx, form part of the initial antioxidant response to O{sub 3}, SO{sub 2}, and UV-B in Nicotiana. 57 refs., 4 figs.

  14. Temporal and Spatial Resolution of Activated Plant Defense Responses in Leaves of Nicotiana benthamiana Infected with Dickeya dadantii

    PubMed Central

    Pérez-Bueno, María L.; Granum, Espen; Pineda, Mónica; Flors, Víctor; Rodriguez-Palenzuela, Pablo; López-Solanilla, Emilia; Barón, Matilde

    2016-01-01

    The necrotrophic bacteria Dickeya dadantii is the causal agent of soft-rot disease in a broad range of hosts. The model plant Nicotiana benthamiana, commonly used as experimental host for a very broad range of plant pathogens, is susceptible to infection by D. dadantii. The inoculation with D. dadantii at high dose seems to overcome the plant defense capacity, inducing maceration and death of the tissue, although restricted to the infiltrated area. By contrast, the output of the defense response to low dose inoculation is inhibition of maceration and limitation in the growth, or even eradication, of bacteria. Responses of tissue invaded by bacteria (neighboring the infiltrated areas after 2–3 days post-inoculation) included: (i) inhibition of photosynthesis in terms of photosystem II efficiency; (ii) activation of energy dissipation as non-photochemical quenching in photosystem II, which is related to the activation of plant defense mechanisms; and (iii) accumulation of secondary metabolites in cell walls of the epidermis (lignins) and the apoplast of the mesophyll (phytoalexins). Infiltrated tissues showed an increase in the content of the main hormones regulating stress responses, including abscisic acid, jasmonic acid, and salicylic acid. We propose a mechanism involving the three hormones by which N. benthamiana could activate an efficient defense response against D. dadantii. PMID:26779238

  15. Analysis of the antioxidant response of Nicotiana benthamiana to infection with two strains of Pepper mild mottle virus

    PubMed Central

    Hakmaoui, A.; Pérez-Bueno, M. L.; Barón, M.

    2012-01-01

    The present study was carried out to investigate the role of reactive oxygen species (ROS) metabolism in symptom development and pathogenesis in Nicotiana benthamiana plants upon infection with two strains of Pepper mild mottle virus, the Italian (PMMoV-I) and the Spanish (PMMoV-S) strains. In this host, it has been shown that PMMoV-I is less virulent and plants show the capability to recover 21 d after inoculation. Analyses of oxidative stress biomarkers, ROS-scavenging enzyme activities, and antioxidant compounds were conducted in plants at different post-infection times. Only PMMoV-I stimulated a defence response through: (i) up-regulation of different superoxide dismutase isozymes; (ii) maintenance of adequate levels of three peroxiredoxins (2-Cys Prx, Prx IIC, and Prx IIF); and (iii) adjustments in the glutathione pool to maintain the total glutathione content. Moreover, there was an increase in the level of oxidized glutathione and ascorbate in the recovery phase of PMMoV-I-infected plants. The antioxidant response and the extent of oxidative stress in N. benthamiana plants correlates to: (i) the severity of the symptoms elicited by either strain of PMMoV; and (ii) the high capacity of PMMoV-I-infected plants for symptom recovery and delayed senescence, compared with PMMoV-S-infected plants. PMID:22915745

  16. Deletion of a MFS transporter-like gene in Cercospora nicotianae reduces cercosporin toxin accumulation and fungal virulence.

    PubMed

    Choquer, Mathias; Lee, Miin-Huey; Bau, Huey-Jiunn; Chung, Kuang-Ren

    2007-02-01

    Many phytopathogenic Cercospora species produce a host-nonselective polyketide toxin, called cercosporin, whose toxicity exclusively relies on the generation of reactive oxygen species. Here, we describe a Cercospora nicotianae CTB4 gene that encodes a putative membrane transporter and provide genetic evidence to support its role in cercosporin accumulation. The predicted CTB4 polypeptide has 12 transmembrane segments with four conserved motifs and has considerable similarity to a wide range of transporters belonging to the major facilitator superfamily (MFS). Disruption of the CTB4 gene resulted in a mutant that displayed a drastic reduction of cercosporin production and accumulation of an unknown brown pigment. Cercosporin was detected largely from fungal hyphae of ctb4 disruptants, but not from the surrounding medium, suggesting that the mutants were defective in both cercosporin biosynthesis and secretion. Cercosporin purified from the ctb4 disruptants exhibited toxicity to tobacco suspension cells, insignificantly different from wild-type, whereas the disruptants formed fewer lesions on tobacco leaves. The ctb4 null mutants retained normal resistance to cercosporin and other singlet oxygen-generating photosensitizers, indistinguishable from the parental strain. Transformation of a functional CTB4 clone into a ctb4 null mutant fully revived cercosporin production. Thus, we propose that the CTB4 gene encodes a putative MFS transporter responsible for secretion and accumulation of cercosporin.

  17. Molecular characterization of Pvr9 that confers a hypersensitive response to Pepper mottle virus (a potyvirus) in Nicotiana benthamiana.

    PubMed

    Tran, Phu-Tri; Choi, Hoseong; Choi, Doil; Kim, Kook-Hyung

    2015-07-01

    There are some R genes against potyviruses which were mapped in pepper. However, none of them has been characterized at the molecular level. In this study, we characterized Pvr9 which is an Rpi-blb2 ortholog from pepper and confers a hypersensitive response to Pepper mottle virus (PepMoV) in a transient expression system in Nicotiana benthamiana. This gene putatively encoded for 1298 amino acids and is located on pepper chromosome 6. PepMoV NIb was the elicitor of the Pvr9-mediated hypersensitive response. NIb from several other potyviruses also elicited the hypersensitive response. Inoculation of pepper with PepMoV resulted in a minor increase in Pvr9 transcription in the resistant cultivar CM334 and a slight down-regulation in the susceptible cultivar Floral Gem. The 5' upstream region of Pvr9 from cultivar CM334 had higher transcription activity than the region from cultivar Floral Gem. The cultivars CM334 and Floral Gem had non-functional Pvr9 homologs with loss-of-function mutations.

  18. Pretreatment with alternation of light/dark periods improves the tolerance of tobacco (Nicotiana tabacum) to clomazone herbicide.

    PubMed

    Darwish, Majd; Lopez-Lauri, Félicie; El Maataoui, Mohamed; Urban, Laurent; Sallanon, Huguette

    2014-05-01

    This work analyses the effects of alternation of light/dark periods pretreatment (AL) in tobacco plantlets (Nicotiana tabacum L. cv.Virginie vk51) growing in solution with low concentration of the clomazone herbicide. The experimentation has been carried out by exposing the plantlets to successive and regulated periods of light (16min light/8min dark cycles, PAR 50μmolm(-2)s(-1)) for three days. The photosynthesis efficiency was determined by mean of the chlorophyll fluorescence and JIP-test. The AL pretreatment improved the clomazone tolerance; this has been observed by the increase in the leaf area of the plant, the maximal photochemical quantum efficiency of PSII (Fv/Fm), the actual PSII efficiency (ФPSII), the performance index (PIabs), the electron flux beyond Quinone A (1-VJ), and also by the diminution of the energy dissipating into heat (DI0/RC). Furthermore, AL pretreatment led to low accumulation of hydrogen peroxide (H2O2) which proves that the scavenging enzymatic system have been activated before clomazone treatment. In the plantlets pretreated with AL, with regard to the ascorbate content, some of antioxidant enzyme whose function is associated with it have continued to scavenge reactive oxygen species (ROS) induced by clomazone, such as ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). So, the observed photooxidative damages induced by clomazone herbicide were noticeably reduced.

  19. Effects of plant vascular architecture on aboveground-belowground-induced responses to foliar and root herbivores on Nicotiana tabacum.

    PubMed

    Kaplan, Ian; Halitschke, Rayko; Kessler, André; Sardanelli, Sandra; Denno, Robert F

    2008-10-01

    Herbivores induce systemic changes in plant traits, and the strength of these induced responses is often associated with the degree of vascular connectivity that links damaged and undamaged plant tissues. Although this phenomenon is known to occur aboveground in leaves, it is unknown whether or not leaf-root induction similarly follows the vascular architecture of plants. To test for this possibility, we manipulated foliar and root herbivory on tobacco (Nicotiana tabacum) by the leaf-chewing insect Spodoptera exigua and the root-galling nematode Meloidogyne incognita. Subsequent changes in secondary chemistry (alkaloids and phenolics) were measured in leaves and roots that were orthostichous (vertically aligned) and nonorthostichous (opposite) from the herbivore-damaged tissues. Aboveground caterpillar herbivory elicited stronger secondary chemical responses in orthostichous compared with nonorthostichous plant tissues, although the magnitude of this difference was greater in leaves than roots. However, belowground nematode herbivory did not affect the secondary chemistry of tobacco leaves, despite inducing strong local responses in roots. Thus, plant vascular architecture can mediate the magnitude of systemic induction in roots as well as in leaves, with stronger responses in tissues that are more closely aligned. As a result, herbivores that co-occur on the same sector of plant (both aboveground and belowground) may be more likely to affect one another via induced responses than herbivores that occur on plant tissues sharing fewer resources.

  20. Role of Rice Stripe Virus NSvc4 in Cell-to-Cell Movement and Symptom Development in Nicotiana benthamiana.

    PubMed

    Xu, Yi; Zhou, Xueping

    2012-01-01

    Our previous work has demonstrated that the NSvc4 protein of Rice stripe virus (RSV) functions as a cell-to-cell movement protein. However, the mechanisms whereby RSV traffics through plasmodesmata (PD) are unknown. Here we provide evidence that the NSvc4 moves on the actin filament and endoplasmic reticulum network, but not microtubules, to reach cell wall PD. Disruption of cytoskeleton using different inhibitors altered NSvc4 localization to PD, thus impeding RSV infection of Nicotiana benthamiana. Sequence analyses and deletion mutagenesis experiment revealed that the N-terminal 125 amino acids (AAs) of the NSvc4 determine PD targeting and that a transmembrane domain spanning AAs 106-125 is critical for PD localization. We also found that the NSvc4 protein can localize to chloroplasts in infected cells. Analyses using deletion mutants revealed that the N-terminal 73 AAs are essential for chloroplast localization. Furthermore, expression of NSvc4 from a Potato virus X (PVX) vector resulted in more severe disease symptoms than PVX alone in systemically infected N. benthamiana leaves. Expression of NSvc4 in Spodoptera frugiperda 9 cells did not elicit tubule formation, but instead resulted in punctate foci at the plasma membrane. These findings shed new light on our understanding of the movement mechanisms whereby RSV infects host plants. PMID:23233857

  1. Overexpression of Cotton GhMPK11 Decreases Disease Resistance through the Gibberellin Signaling Pathway in Transgenic Nicotiana benthamiana.

    PubMed

    Wang, Fang; Wang, Chen; Yan, Yan; Jia, Haihong; Guo, Xingqi

    2016-01-01

    Many changes in development, growth, hormone activity and environmental stimuli responses are mediated by mitogen-activated protein kinase (MAPK) cascades. However, in plants, studies on MAPKs have mainly focused on MPK3, MPK4 and MPK6. Here, a novel group B MAPK gene, GhMPK11, was isolated from cotton (Gossypium hirsutum L.) and characterized. Both promoter and expression pattern analyses revealed that GhMPK11 is involved in defense responses and signaling pathways. GhMPK11 overexpression in Nicotiana benthamiana plants could increase gibberellin 3 (GA3) content through the regulation of GA-related genes. Interestingly, either GhMPK11 overexpression or exogenous GA3 treatment in N. benthamiana plants could enhance the susceptibility of these plants to the infectious pathogens Ralstonia solanacearum and Rhizoctonia solani. Moreover, reactive oxygen species (ROS) accumulation was increased after pathogen infiltration due to the increased expression of ROS-related gene respiratory burst oxidative homologs (RbohB) and the decreased expression or activity of ROS detoxification enzymes regulated by GA3, such as superoxide dismutases (SODs), peroxidases (PODs), catalase (CAT) and glutathione S-transferase (GST). Taken together, these results suggest that GhMPK11 overexpression could enhance the susceptibility of tobacco to pathogen infection through the GA3 signaling pathway via down-regulation of ROS detoxification enzymes. PMID:27242882

  2. Strategies for the selection and characterization of aluminum-resistant variants from cell cultures of Nicotiana plumbaginifolia.

    PubMed

    Conner, A J; Meredith, C P

    1985-12-01

    The development of strategies for selecting and characterizing aluminum-resistant variants from Nicotiana plumbaginifolia Viv. cell cultures is described. Plated cells, smeared callus, in-vitro-grown shoots, and seedlings of wild-type N. plumbaginifolia all showed similar responses to Al, with total growth inhibition at or above 600 μM Al. The strict control of both cell density and aggregate size is important in selection experiments for total inhibition of the growth of wild-type cells. Two approaches for the selection of Al-resistant variants were used. In a direct method, cells were plated onto medium containing 600 μM Al which inhibited growth and chlorophyll synthesis in wildtype cells. A double selection strategy based on both cell growth and greening was used to isolate 29 Al-resistant variants. In the other approach, a rescue method, suspensions were cultured for 10 d in medium containing 600 μM Al, then plated onto standard medium for recovery of survivors. Using this strategy, 217 Al-resistant variants were selected. After six to twelve weeks of growth in the absence of Al, each variant was cloned and reselected from single cells. Al resistance was retained in 31% and 51% of the variants selected by the direct and rescue strategies, respectively. Seedling segregation data are presented for the progeny (selfed and backcrossed) of plants regenerated from one of the variants and are consistent with those expected for a single dominant mutation.

  3. Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

    PubMed Central

    Hérouart, D; Van Montagu, M; Inzé, D

    1994-01-01

    Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the beta-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS staining was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol. PMID:8165260

  4. Mitochondrial alternative oxidase is involved in both compatible and incompatible host-virus combinations in Nicotiana benthamiana.

    PubMed

    Zhu, Feng; Deng, Xing-Guang; Xu, Fei; Jian, Wei; Peng, Xing-Ji; Zhu, Tong; Xi, De-Hui; Lin, Hong-Hui

    2015-10-01

    The alternative oxidase (AOX) functions in the resistance to biotic stress. However, the mechanisms of AOX in the systemic antiviral defense response and N (a typical resistance gene)-mediated resistance to Tobacco mosaic virus (TMV) are elusive. A chemical approach was undertaken to investigate the role of NbAOX in the systemic resistance to RNA viruses. Furthermore, we used a virus-induced gene-silencing (VIGS)-based genetics approach to investigate the function of AOX in the N-mediated resistance to TMV. The inoculation of virus significantly increased the NbAOX transcript and protein levels and the cyanide-resistant respiration in the upper un-inoculated leaves. Pretreatment with potassium cyanide greatly increased the plant's systemic resistance, whereas the application of salicylhydroxamic acid significantly compromised the plant's systemic resistance. Additionally, in NbAOX1a-silenced N-transgenic Nicotiana benthamiana plants, the inoculated leaf collapsed and the movement of TMV into the systemic tissue eventually led to the spreading of HR-PCD and the death of the whole plant. The hypersensitive response marker gene HIN1 was significantly increased in the NbAOX1a-silenced plants. Significant amounts of TMV-CP mRNA and protein were detected in the NbAOX1a-silenced plants but not in the control plants. Overall, evidence is provided that AOX plays important roles in both compatible and incompatible plant-virus combinations. PMID:26398788

  5. A viral satellite DNA vector-induced transcriptional gene silencing via DNA methylation of gene promoter in Nicotiana benthamiana.

    PubMed

    Ju, Zheng; Wang, Lei; Cao, Dongyan; Zuo, Jinhua; Zhu, Hongliang; Fu, Daqi; Luo, Yunbo; Zhu, Benzhong

    2016-09-01

    Virus-induced gene silencing (VIGS) has been widely used for plant functional genomics study at the post-transcriptional level using various DNA or RNA viral vectors. However, while virus-induced transcriptional gene silencing (VITGS) via DNA methylation of gene promoter was achieved using several plant RNA viral vectors, it has not yet been done using a satellite DNA viral vector. In this study, a viral satellite DNA associated with tomato yellow leaf curl China virus (TYLCCNV), which has been modified as a VIGS vector in previous research, was developed as a VITGS vector. Firstly, the viral satellite DNA VIGS vector was further optimized to a more convenient p1.7A+2mβ vector with high silencing efficiency of the phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. Secondly, the constructed VITGS vector (TYLCCNV:35S), which carried a portion of the cauliflower mosaic virus 35S promoter, could successfully induce heritable transcriptional gene silencing (TGS) of the green fluorescent protein (GFP) gene in the 35S-GFP transgenic N. benthamiana line 16c plants. Moreover, bisulfite sequencing results revealed higher methylated cytosine residues at CG, CHG and CHH sites of the 35S promoter sequence in TYLCCNV:35S-inoculated plants than in TYLCCNV-inoculated line 16c plants (control). Overall, these results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes. PMID:27422476

  6. Transgenic down-regulation of ARGONAUTE2 expression in Nicotiana benthamiana interferes with several layers of antiviral defenses.

    PubMed

    Odokonyero, Denis; Mendoza, Maria R; Alvarado, Veria Y; Zhang, Jiantao; Wang, Xiaofeng; Scholthof, Herman B

    2015-12-01

    The present study aimed to analyze the contribution of Nicotiana benthamiana ARGONAUTE2 (NbAGO2) to its antiviral response against different viruses. For this purpose, dsRNA hairpin technology was used to reduce NbAGO2 expression in transgenic plants as verified with RT-PCR. This reduction was specific because the expression of other NbAGOs was not affected, and did not cause obvious developmental defects under normal growth conditions. Inoculation of transgenic plants with an otherwise silencing-sensitive GFP-expressing Tomato bushy stunt virus (TBSV) variant resulted in high GFP accumulation because antiviral silencing was compromised. These transgenic plants also exhibited accelerated spread and/or enhanced susceptibility and symptoms for TBSV mutants defective for P19 or coat protein expression, other tombusviruses, Tobacco mosaic virus, and Potato virus X; but not noticeably for Foxtail mosaic virus. These findings support the notion that NbAGO2 in N. benthamiana can contribute to antiviral defense at different levels. PMID:26454664

  7. Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana.

    PubMed

    Rajput, Nasir Ahmed; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Zhang, Qimeng; Ru, Yanyan; Sun, Peng; Dou, Daolong

    2015-12-01

    The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses.

  8. Isoprene emission aids recovery of photosynthetic performance in transgenic Nicotiana tabacum following high intensity acute UV-B exposure.

    PubMed

    Centritto, Mauro; Haworth, Matthew; Marino, Giovanni; Pallozzi, Emanuele; Tsonev, Tsonko; Velikova, Violeta; Nogues, Isabel; Loreto, Francesco

    2014-09-01

    Isoprene emission by terrestrial plants is believed to play a role in mitigating the effects of abiotic stress on photosynthesis. Ultraviolet-B light (UV-B) induces damage to the photosynthetic apparatus of plants, but the role of isoprene in UV-B tolerance is poorly understood. To investigate this putative protective role, we exposed non-emitting (NE) control and transgenic isoprene emitting (IE) Nicotiana tabacum (tobacco) plants to high intensity UV-B exposure. Methanol emissions increased with UV-B intensity, indicating oxidative damage. However, isoprene emission was unaffected during exposure to UV-B radiation, but declined in the 48 h following UV-B treatment at the highest UV-B intensities of 9 and 15 Wm(-2). Photosynthesis and the performance of photosystem II (PSII) declined to similar extents in IE and NE plants following UV-B exposure, suggesting that isoprene emission did not ameliorate the immediate impact of UV-B on photosynthesis. However, after the stress, photosynthesis and PSII recovered in IE plants, which maintained isoprene formation, but not in NE plants. Recovery of IE plants was also associated with elevated antioxidant levels and cycling; suggesting that both isoprene formation and antioxidant systems contributed to reinstating the integrity and functionality of cellular membranes and photosynthesis following exposure to excessive levels of UV-B radiation.

  9. The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

    PubMed Central

    Blackman, LM; Boevink, P; Cruz, SS; Palukaitis, P; Oparka, KJ

    1998-01-01

    The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer. PMID:9548980

  10. NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development

    PubMed Central

    Zhang, Xiao-mei; Wang, Ying; Lv, Xiao-meng; Li, Hui; Sun, Peng; Lu, Hai; Li, Feng-lan

    2009-01-01

    Proteinases play a critical role in developmental homeostasis and in response to environ-mental stimuli. Our present research reports that a new cysteine protease, NtCP56, is involved in the development of pollen grains in Nicotiana tabacum L. The NtCP56 gene, which encodes a protein of 361 amino acid residues with a calculated molecular mass of 40 kDa, is strongly expressed in anthers. The recombinant NtCP56 showed a high activity towards casein. Kinetic analysis revealed a Km of 2.20 mg ml−1 and Vmax of 11.07 μg ml−1 min−1. The recombinant NtCP56 retained more than 50% of its maximum enzymatic activity from 20 °C to 60 °C with an optimum Tm range of 30–50 °C. The enzyme had a maximum activity at approximately pH 6.5. Suppression of the NtCP56 gene in anti-sense transgenic tobaccos resulted in the sterility of pollen grains. Our data indicated that, as a cysteine protease, NtCP56 might play an important role in pollen development. PMID:19246592

  11. The dihydrolipoyl acyltransferase gene BCE2 participates in basal resistance against Phytophthora infestans in potato and Nicotiana benthamiana.

    PubMed

    Wang, Hongyang; Sun, Chunlian; Jiang, Rui; He, Qin; Yang, Yu; Tian, Zhejuan; Tian, Zhendong; Xie, Conghua

    2014-07-01

    Dihydrolipoyl acyltransferase (EC 2.3.1.12), a branched-chain α-ketoacid dehydrogenase E2 subunit (BCE2), catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. However, the role of BCE2 responding to biotic stress in plant is not clear. In this study, we cloned and characterized a BCE2 gene from potato, namely StBCE2, which was previously suggested to be involved in Phytophthora infestans-potato interaction. We found that the expression of StBCE2 was strongly induced by both P. infestans isolate HB09-14-2 and salicylic acid. Besides, when the homolog of StBCE2 in Nicotiana benthamiana named NbBCE2 was silenced, plants showed increased susceptibility to P. infestans and reduced accumulation of hydrogen peroxide (H2O2). Furthermore, we found that a marker gene NbrbohB involved in the production of reactive oxygen species, was also suppressed in NbBCE2-silenced plants. However, silencing of NbBCE2 had no significant effect on the hypersensitive responses trigged by INF1, R3a-AVR3a(KI) pair or Rpi-vnt1.1-AVR-vnt1.1 pair. Our results suggest that BCE2 is associated with the basal resistance to P. infestans by regulating H2O2 production. PMID:24913048

  12. Overexpression of Cotton GhMPK11 Decreases Disease Resistance through the Gibberellin Signaling Pathway in Transgenic Nicotiana benthamiana

    PubMed Central

    Wang, Fang; Wang, Chen; Yan, Yan; Jia, Haihong; Guo, Xingqi

    2016-01-01

    Many changes in development, growth, hormone activity and environmental stimuli responses are mediated by mitogen-activated protein kinase (MAPK) cascades. However, in plants, studies on MAPKs have mainly focused on MPK3, MPK4 and MPK6. Here, a novel group B MAPK gene, GhMPK11, was isolated from cotton (Gossypium hirsutum L.) and characterized. Both promoter and expression pattern analyses revealed that GhMPK11 is involved in defense responses and signaling pathways. GhMPK11 overexpression in Nicotiana benthamiana plants could increase gibberellin 3 (GA3) content through the regulation of GA-related genes. Interestingly, either GhMPK11 overexpression or exogenous GA3 treatment in N. benthamiana plants could enhance the susceptibility of these plants to the infectious pathogens Ralstonia solanacearum and Rhizoctonia solani. Moreover, reactive oxygen species (ROS) accumulation was increased after pathogen infiltration due to the increased expression of ROS-related gene respiratory burst oxidative homologs (RbohB) and the decreased expression or activity of ROS detoxification enzymes regulated by GA3, such as superoxide dismutases (SODs), peroxidases (PODs), catalase (CAT) and glutathione S-transferase (GST). Taken together, these results suggest that GhMPK11 overexpression could enhance the susceptibility of tobacco to pathogen infection through the GA3 signaling pathway via down-regulation of ROS detoxification enzymes. PMID:27242882

  13. Analysis of Guard Cell Viability and Action in Senescing Leaves of Nicotiana glauca (Graham), Tree Tobacco 1

    PubMed Central

    Ozuna, Richard; Yera, Ramon; Ortega, Kim; Tallman, Gary

    1985-01-01

    In an attempt to determine whether low epidermal conductances to water vapor diffusion of senescing leaves were caused by internal changes in guard cells or by factors external to guard cells, stomatal behavior was examined in intact senescing and nonsenescing leaves of Nicotiana glauca (Graham), tree tobacco, grown in the field or in an environmental chamber. Conductances of senescing leaves were 5 to 10% of the maximum conductances of nonsenescing leaves of the same plant, yet guard cell duplexes isolated from epidermal peels of senescing leaves developed full turgor in the light in solutions containing KCl, and sodium cobaltinitrite staining showed that K+ accumulated as turgor developed. Ninety-five per cent of the guard cells isolated from senescing leaves concentrated neutral red and excluded trypan blue. Intercellular leaf CO2 concentrations of senescing and nonsenescing leaves of chamber-grown plants were not significantly different (about 240 microliters per liter), but the potassium contents of adaxial and abaxial epidermes of senescing leaves taken from plants grown in the field were less than half those of nonsenescing leaves. We conclude that guard cells do not undergo the orderly senescence process that characteristically takes place in mesophyll tissue during whole-leaf senescence and that the reduced conductances of senescing leaves are produced by factors external to guard cells. PMID:16664404

  14. Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses

    PubMed Central

    Mathew, Lolita George; Herbst-Kralovetz, Melissa M.; Mason, Hugh S.

    2014-01-01

    Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs. PMID:24949472

  15. Severely reduced gravitropism in dark-grown hypocotyls of a starch-deficient mutant of Nicotiana sylvestris

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Sack, F. D.

    1990-01-01

    Gravitropism in dark-grown hypocotyls of the wild type was compared with a starch-deficient Nicotiana sylvestris mutant (NS 458) to test the effects of starch deficiency on gravity sensing. In a time course of curvature measured using infrared video, the response of the mutant was greatly reduced compared to the wild type; 72 hours after reorientation, curvature was about 10 degrees for NS 458 and about 70 degrees for wild type. In dishes maintained in a vertical orientation, wild-type hypocotyls were predominantly vertical, whereas NS 458 hypocotyls were severely disoriented with about 5 times more orientational variability than wild type. Since the growth rates were equal for both genotypes and phototropic curvature was only slightly inhibited in NS 458, the mutation probably affects gravity perception rather than differential growth. Our data suggest that starch deficiency reduces gravitropic sensitivity more in dark-grown hypocotyls than in dark- or light-grown roots in this mutant and support the hypothesis that amyloplasts function as statoliths in shoots as well as roots.

  16. Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of phytoremediation of polychlorinated biphenyls

    PubMed Central

    Novakova, Martina; Mackova, Martina; Antosova, Zuzana; Viktorova, Jitka; Szekeres, Miklos; Demnerova, Katerina

    2010-01-01

    The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for β-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied. PMID:21468210

  17. A pleiotropic drug resistance transporter in Nicotiana tabacum is involved in defense against the herbivore Manduca sexta.

    PubMed

    Bienert, Manuela D; Siegmund, Stephanie E G; Drozak, Anna; Trombik, Tomasz; Bultreys, Alain; Baldwin, Ian T; Boutry, Marc

    2012-12-01

    Pleiotropic drug resistance (PDR) transporters are a group of membrane proteins belonging to the ABCG sub-family of ATP binding cassette (ABC) transporters. There is clear evidence for the involvement of plant ABC transporters in resistance to fungal and bacterial pathogens, but not in the biotic stress response to insect or herbivore attack. Here, we describe a PDR transporter, ABCG5/PDR5, from Nicotiana tabacum. GFP fusion and subcellular fractionation studies revealed that ABCG5/PDR5 is localized to the plasma membrane. Staining of transgenic plants expressing the GUS reporter gene under the control of the ABCG5/PDR5 transcription promoter and immunoblotting of wild-type plants showed that, under standard growth conditions, ABCG5/PDR5 is highly expressed in roots, stems and flowers, but is only expressed at marginal levels in leaves. Interestingly, ABCG5/PDR5 expression is induced in leaves by methyl jasmonate, wounding, pathogen infiltration, or herbivory by Manduca sexta. To address the physiological role of ABCG5/PDR5, N. tabacum plants silenced for the expression of ABCG5/PDR5 were obtained. No phenotypic modification was observed under standard conditions. However, a small increase in susceptibility to the fungus Fusarium oxysporum was observed. A stronger effect was observed in relation to herbivory: silenced plants allowed better growth and faster development of M. sexta larvae than wild-type plants, indicating an involvement of this PDR transporter in resistance to M. sexta herbivory.

  18. WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana[OPEN

    PubMed Central

    Adachi, Hiroaki; Nakano, Takaaki; Miyagawa, Noriko; Ishihama, Nobuaki; Yoshioka, Miki; Katou, Yuri; Yaeno, Takashi

    2015-01-01

    Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB. PMID:26373453

  19. Phytoplasma SAP11 alters 3-isobutyl-2-methoxypyrazine biosynthesis in Nicotiana benthamiana by suppressing NbOMT1.

    PubMed

    Tan, Choon Meng; Li, Chia-Hua; Tsao, Nai-Wen; Su, Li-Wen; Lu, Yen-Ting; Chang, Shu Heng; Lin, Yi Yu; Liou, Jyun-Cyuan; Hsieh, Li-Ching; Yu, Jih-Zu; Sheue, Chiou-Rong; Wang, Sheng-Yang; Lee, Chin-Fa; Yang, Jun-Yi

    2016-07-01

    Phytoplasmas are bacterial phytopathogens that release virulence effectors into sieve cells and act systemically to affect the physiological and morphological state of host plants to promote successful pathogenesis. We show here that transgenic Nicotiana benthamiana lines expressing the secreted effector SAP11 from Candidatus Phytoplasma mali exhibit an altered aroma phenotype. This phenomenon is correlated with defects in the development of glandular trichomes and the biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP). IBMP is a volatile organic compound (VOC) synthesized by an O-methyltransferase, via a methylation step, from a non-volatile precursor, 3-isobutyl-2-hydroxypyrazine (IBHP). Based on comparative and functional genomics analyses, NbOMT1, which encodes an O-methyltransferase, was found to be highly suppressed in SAP11-transgenic plants. We further silenced NbOMT1 through virus-induced gene silencing and demonstrated that this enzyme influenced the accumulation of IBMP in N. benthamiana In vitro biochemical analyses also showed that NbOMT1 can catalyse IBHP O-methylation in the presence of S-adenosyl-L-methionine. Our study suggests that the phytoplasma effector SAP11 has the ability to modulate host VOC emissions. In addition, we also demonstrated that SAP11 destabilized TCP transcription factors and suppressed jasmonic acid responses in N. benthamiana These findings provide valuable insights into understanding how phytoplasma effectors influence plant volatiles. PMID:27279277

  20. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

    PubMed

    Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2009-02-01

    The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

  1. Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene.

    PubMed

    Fuoco, Roger; Bogani, Patrizia; Capodaglio, Gabriele; Del Bubba, Massimo; Abollino, Ornella; Giannarelli, Stefania; Spiriti, Maria Michela; Muscatello, Beatrice; Doumett, Saer; Turetta, Clara; Zangrando, Roberta; Zelano, Vincenzo; Buiatti, Marcello

    2013-05-01

    Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plant's hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.

  2. Resistance to tomato yellow leaf curl geminivirus in Nicotiana benthamiana plants transformed with a truncated viral C1 gene.

    PubMed

    Noris, E; Accotto, G P; Tavazza, R; Brunetti, A; Crespi, S; Tavazza, M

    1996-10-01

    The C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a multifunctional protein (Rep) involved in replication. A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (aa) of the Rep protein, was cloned under the control of the CaMV 35S promoter and introduced into Nicotiana benthamiana using Agrobacterium tumefaciens. The same sequence was also cloned in antisense orientation. When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines. Two such lines were further studied. The presence of the transgene was verified and its expression was followed at intervals. All plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of transgenic mRNA and protein at the time of infection. Resistance was overcome in a few plants at 9 wpi, and in most at 15 wpi. Infection of leaf discs derived from transgenic plants showed that expression of the transgene correlated with a substantial reduction of viral DNA replication. Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep protein and suggested that the small ORF C4, also present in our construct, plays no role in the resistance observed. The results obtained using both transient and stable gene expression systems show that the expression of the N-terminal 210 aa of the TYLCV Rep protein efficiently interferes with virus infection. PMID:8862407

  3. Synthesis of glycolate from pyruvate via isocitrate lyase by tobacco leaves in light. [Nicotiana tabacum var Havana Seed

    SciTech Connect

    Zelitch, I. )

    1988-02-01

    Tobacco (Nicotiana tabacum var Havana Seed) leaf discs were supplied tracer quantities of (2-{sup 14}C)- and (3-{sup 14}C) pyruvate for 60 minutes in steady state photosynthesis with 21% or 1% O{sub 2}, and the glycolate oxidase inhibitor {alpha}-hydroxy-2-pyridinemethanesulfonic acid was then added for 5 or 10 minutes to cause glycolate to accumulate. The (3-{sup 14}C) pyruvate was converted directly to glycolate as shown by a 50% greater than equal-labeled {sup 14}C in C-2 of glycolate, and the fraction of {sup 14}C in C-2 increased in 1% O{sub 2} to 80% greater than equal-labeled. This suggests the pathway using pyruvate is less O{sub 2}-dependent than the oxygenase reaction producing glycolate from the Calvin cycle. The formation of glycolate from pyruvate in the leaf discs was time-dependent and with (2-{sup 14}C)- and (3-{sup 14}C) pyruvate supplied leaf discs the C-2 of glyoxylate derived from C-2 of isocitrate was labeled asymmetrically in a manner similar to the asymmetrical labeling of C-2 of glycolate under a number of conditions. Thus glycolate was probably formed by the reduction of glyoxylate. Isocitric lyase activity of tobacco leaves was associated with leaf mitochondria, through most of the activity was in the supernatant fraction after differential centrifugation of leaf homogenates.

  4. DEVELOPMENT AND ASSESSMENT OF EST-SSR MARKER FOR THE GENETIC DIVERSITY AMONG TOBACCOS (Nicotiana tabacum L.).

    PubMed

    Cai, C; Yang, Y; Cheng, L; Tong, C; Feng, J

    2015-06-01

    Because of the advantages of EST-SSR markers, it has been employed as powerful markers for genetic diversity analysis, comparative mapping and phylogenetic studies. In this study, a total of 429,869 tobacco (Nicotiana tabacum L.) ESTs were downloaded from the public databases, which offers an opportunity to identify SSRs in ESTs by data mining, and 38,165 SSRs were identified from 379,967 uni-ESTs with the frequency of one SSR per 5.52 kb. Mono- and tri-nucleotide repeat motifs were the dominant repeat types, accounting for 40.53 and 34.51% of all SSRs, respectively. After eliminating mononucleotide-containing sequences, 86 pairs of primers were designed to amplify in four tobacco accessions. Only 15 primers (17.44%) showed polymorphism, and then they were further used to assess genetic diversity of 20 tobacco accessions. Unweighted pair-group method with arithmetic average dendrograms (UPGMA) and principal coordinates analysis plots (PCA) revealed genetic differentiation between N. rustica and N. tabacum, and between oriental tobacco and other accessions of N. tabacum. The present study reported the development of EST-SSR markers in tobacco by exploiting EST databases, and confirmed the effective way to develop markers. These EST-SSRs can serve in studies on cultivar identification, genetic diversity analysis, and genetics in tobacco.

  5. Early inhibition of photosynthesis during development of Mn toxicity in tobacco. [Nicotiana tabacum L. cv KY14

    SciTech Connect

    Nable, R.O.; Houtz, R.L.; Cheniae, G.M. )

    1988-04-01

    Early physiological effects of developing Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) were examined in glass-house/water cultured plants grown at high (summer) and low (winter) photon flux. Following transfer of plants to solutions containing 1 millimolar Mn{sup 2+}, sequential samplings were made at various times for the following 9 days, during which Mn accumulation by leaves increased rapidly from {approx} 70 on day 0 to {approx} 1700 and {approx} 5000 microgram per gram dry matter after 1 and 9 days, respectively. In plants grown at high photon flux, net photosynthesis declined by {approx} 20 and {approx} 60% after 1 and 9 days, respectively, and the onset of this decline preceded appearance (after 3 to 4 days) of visible foliar symptoms of Mn toxicity. Intercellular CO{sub 2} concentrations and rates of transpiration were not significantly affected. Though the activity of latent or activated polyphenol oxidase increased in parallel with Mn accumulation, neither leaf respiration nor the activity of catalase (EC 1.11.1.6) and peroxidase (EC 1.10.1.7) were greatly affected. These effects from Mn toxicity could not be explained by any changes in protein or chlorophyll abundance. Additionally, they were not a consequence of Mn induced Fe deficiency. Therefore, inhibition of net photosynthesis and enhancement of polyphenol oxidase activity are early indicators of excess Mn accumulation in tobacco leaves.

  6. [The role of Cd-binding proteins and phytochelatins in the formation of cadmium resistance in Nicotiana plumbaginifolia cell lines].

    PubMed

    Fenik, S I; Solodushko, V G; Kaliniak, T B; Blium, Ia B

    2007-01-01

    Nicotiana plumbaginifolia callus lines with the equal resistance to cadmium have been produced under different selective conditions--either without inhibition of the phytochelatin synthesis (line Cd-R) or in the presence of the inhibitor butionine sulfoximine (line Cd-Ri). The level of phytochelatin synthesis in the line Cd-R five-fold exceeded the control value and in the line Cd-Ri it was twice as much as in the control. It was shown that in the control line mainly three cadmium-binding proteins are expressed of the molecular weihgts 41, 34 and 19 kD. The common feature of the both resistant lines is the expression of the cadmium-binding proteins of 40, 37 and 19 kD. The resistant lines differ with respect to the synthesis of relatively low-molecular cadmium-binding proteins. The proteins of the molecular weights 12.5, 11.5 and 9 kD are expressed in the line Cd-R, while the proteins of 13 and 10 kD are expressed in the line Cd-Ri. It was supposed that both the phytochelatins and the Cd-binding proteins contribute to the resisitance of N. plumbaginifolia callus lines to cadmium and the lack of the phytochelatins can be equilibrated by the changes in the low-molecular Cd-binding protein synthesis.

  7. Tobacco (Nicotiana tobaccum) nuclear transgenics with high copy number can express NPTII driven by the chloroplast psbA promoter.

    PubMed

    Ye, G N; Pang, S Z; Sanford, J C

    1996-03-01

    A chloroplast expression vector containing the NPTII gene under the control of apsbA promoter (psbA-NPTII) was constructed, and was biolistically delivered into both suspension cells and leaf strips of tobacco (Nicotiana tabaccum). Analyses of subsequently recovered kanamycin-resistant transgenic plants indicate that the psbA-NPTII gene was not located in the chloroplast, but was in the nucleus in very high copy number. This conclusion was based upon results from: (1) Southern hybridization analyses of chloroplast and nuclear DNAs using NPTII, chloroplast-marker, and nuclear-marker probes; (2) pulse-field gel electrophoresis; and (3) kanamycin screening of sexual progenies. This study suggests that the nuclear expression of the NPTII gene may have been associated with many copies of the psbA-NPTII construction. Very high copy number in the nucleus might either allow NPTII expression from the otherwise inadequate psbA promoter, or might increase the chance of recombining with upstream tobacco regulatory sequences. PMID:24178457

  8. The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

    PubMed

    Limkul, Juthamard; Misaki, Ryo; Kato, Ko; Fujiyama, Kazuhito

    2015-11-01

    Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production. PMID:26475186

  9. Effects and Effectiveness of Two RNAi Constructs for Resistance to Pepper golden mosaic virus in Nicotiana benthamiana Plants

    PubMed Central

    Medina-Hernández, Diana; Rivera-Bustamante, Rafael Francisco; Tenllado, Francisco; Holguín-Peña, Ramón Jaime

    2013-01-01

    ToChLPV and PepGMV are Begomoviruses that have adapted to a wide host range and are able to cause major diseases in agronomic crops. We analyzed the efficacy of induced resistance to PepGMV in Nicotiana benthamiana plants with two constructs: one construct with homologous sequences derived from PepGMV, and the other construct with heterologous sequences derived from ToChLPV. Plants protected with the heterologous construct showed an efficacy to decrease the severity of symptoms of 45%, while plants protected with the homologous construct showed an efficacy of 80%. Plants protected with the heterologous construct showed a reduction of incidence of 42.86%, while the reduction of incidence in plants protected with the homologous construct was 57.15%. The efficacy to decrease viral load was 95.6% in plants protected with the heterologous construct, and 99.56% in plants protected with the homologous construct. We found, in both constructs, up-regulated key components of the RNAi pathway. This demonstrates that the efficacy of the constructs was due to the activation of the gene silencing mechanism, and is reflected in the decrease of viral genome copies, as well as in recovery phenotype. We present evidence that both constructs are functional and can efficiently induce transient resistance against PepGMV infections. This observation guarantees a further exploration as a strategy to control complex Begomovirus diseases in the field. PMID:24287597

  10. Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven, auxin-dependent patterning.

    PubMed

    Durst, Steffen; Nick, Peter; Maisch, Jan

    2013-08-15

    Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP2) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus. PMID:23545293

  11. GC-MS and MALDI-TOF MS profiling of sucrose esters from Nicotiana tabacum and N. rustica.

    PubMed

    Haliński, Łukasz P; Stepnowski, Piotr

    2013-01-01

    Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the first time to the analysis of the sucrose esters from the surface of Nicotiana L. leaves. The profiles obtained for the model plant N. tabacum were similar to those from the gas chromatography-flame ionization detector (GC-FID) analysis. The most reproducible results were obtained using a dihydroxybenzoic acid (DHB) matrix. The main advantage of this method is that crude plant extracts can be analysed without sample clean-up. GC-MS analysis of Aztec tobacco (N. rustica) extracts revealed the presence of three types of sucrose esters. All identified compounds had three C4-C8 acyl chains substituting the glucose moiety, while the fructose part of the molecule was substituted with 0, 1, or 2 acetyl groups. MALDI-TOF MS analysis of the sucrose ester fraction revealed the presence of compounds not eluting from a GC column. Combining the data from both GC-MS and MALDI-TOF MS experiments, we obtained a full sucrose ester profile, which is based on the molecular weight of the compounds and on the number of acyl chains in the molecule. PMID:23923618

  12. Immediate effects of nectar robbing by Palestine sunbirds (Nectarinia osea) on nectar alkaloid concentrations in tree tobacco (Nicotiana glauca).

    PubMed

    Kaczorowski, Rainee L; Koplovich, Avi; Sporer, Frank; Wink, Michael; Markman, Shai

    2014-04-01

    Plant secondary metabolites (PSMs), such as alkaloids, are often found in many parts of a plant, including flowers, providing protection to the plant from various types of herbivores or microbes. PSMs are also present in the floral nectar of many species, but typically at lower concentrations than in other parts of the plant. Nectar robbers often damage floral tissue to access the nectar. By doing so, these nectar robbers may initiate an increase of PSMs in the floral nectar. It is often assumed that it takes at least a few hours before the plant demonstrates an increase in PSMs. Here, we addressed the question of whether PSMs in the floral tissue are immediately being released into the floral nectar following nectar robbing. To address this research question, we investigated whether there was an immediate effect of nectar robbing by the Palestine Sunbird (Nectarinia osea) on the concentration of nectar alkaloids, nicotine and anabasine, in Tree Tobacco (Nicotiana glauca). We found that the concentration of anabasine, but not nicotine, significantly increased in floral nectar immediately following simulated nectar robbing. These findings suggest that nectar robbers could be ingesting greater amounts of PSMs than they would if they visit flowers legitimately. As a consequence, increased consumption of neurotoxic nectar alkaloids or other PSMs could have negative effects on the nectar robber.

  13. Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Giddings, T. H. Jr; Staehelin, L. A.; Sack, F. D.

    1990-01-01

    To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips of Nicotiana and Arabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more numerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) larger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.

  14. Subcellular localization of chorismate-mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris.

    PubMed

    d'Amato, T A; Ganson, R J; Gaines, C G; Jensen, R A

    1984-09-01

    The subcellular locations of two readily discriminated chorismate-mutase (EC 5.4.99.5) isoenzymes from Nicotiana silvestris Speg. et Comes were determined in protoplasts prepared from both leaf tissue and isogenic suspension-cultured cells. Differential centrifugation was used to obtain fractions containing plastids, a mixture of mitochondria and microbodies, and soluble cytosolic proteins. Isoenzyme CM-1 is sensitive to feedback inhibition by L-tyrosine and comprises the major fraction of total chorismate mutase in suspension-cultured cells. Isoenzyme CM-2 is not inhibited by L-tyrosine and its expression is maximal in organismal (leaf) tissue. Isoenzyme CM-1 is located in the plastid compartment since (i) proplastids contained more CM-1 activity than chloroplasts, (ii) both chloroplast and proplastid fractions possessed the tyrosine-sensitive isoenzyme, and (iii) latency determinations on washed chloroplast preparations confirmed the internal location of a tyrosine-sensitive isoenzyme. Isoenzyme CM-2 is located in the cytosol since (i) the supernatant fractions were heavily enriched for the tyrosineinsensitive activity, and (ii) a relatively greater amount of tyrosine-insensitive enzyme was present in the supernatant fraction derived from organismal tissue.

  15. Molecular characterization of NbPAF encoding the alpha6 subunit of the 20S proteasome in Nicotiana benthamiana.

    PubMed

    Kim, Moonil; Yang, Kyoung-Sil; Kim, Yu-Kyung; Paek, Kyung-Hee; Pai, Hyun-Sook

    2003-02-28

    The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex. The NbPAF gene encoding the alpha6 subunit of the 20S proteasome was identified from Nicotiana benthamiana. NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsis, human and yeast. The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome alpha-type subunits signature and nuclear localization signal at the N-terminus. The genomic Southern blot analysis suggests that the N. benthamiana genome contains one copy of NbPAF. The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems, but it was almost undetectable in mature leaves. In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaCl and salicylic acid, but not by abscisic acid and cold treatment in leaves. The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus. PMID:12661772

  16. Theobroxide Treatment Inhibits Wild Fire Disease Occurrence in Nicotiana benthamiana by the Overexpression of Defense-related Genes.

    PubMed

    Ahn, Soon Young; Baek, Kwang-Hyun; Moon, Yong Sun; Yun, Hae Keun

    2013-03-01

    Theobroxide, a novel compound isolated from a fungus Lasiodiplodia theobromae, stimulates potato tuber formation and induces flowering of morning glory by initiating the jasmonic acid synthesis pathway. To elucidate the effect of theobroxide on pathogen resistance in plants, Nicotiana benthamiana plants treated with theobroxide were immediately infiltrated with Pseudomonas syringae pv. tabaci. Exogenous application of theobroxide inhibited development of lesion symptoms, and growth of the bacterial cells was significantly retarded. Semi-quantitative RT-PCRs using the primers of 18 defense-related genes were performed to investigate the molecular mechanisms of resistance. Among the genes, the theobroxide treatment increased the expression of pathogenesis-related protein 1a (PR1a), pathogenesis-related protein 1b (PR1b), glutathione S-transferase (GST), allen oxide cyclase (AOC), and lipoxyganase (LOX). All these data strongly indicate that theobroxide treatment inhibits disease development by faster induction of defense responses, which can be possible by the induction of defense-related genes including PR1a, PR1b, and GST triggered by the elevated jasmonic acid. PMID:25288936

  17. Theobroxide Treatment Inhibits Wild Fire Disease Occurrence in Nicotiana benthamiana by the Overexpression of Defense-related Genes

    PubMed Central

    Ahn, Soon Young; Baek, Kwang-Hyun; Moon, Yong Sun; Yun, Hae Keun

    2013-01-01

    Theobroxide, a novel compound isolated from a fungus Lasiodiplodia theobromae, stimulates potato tuber formation and induces flowering of morning glory by initiating the jasmonic acid synthesis pathway. To elucidate the effect of theobroxide on pathogen resistance in plants, Nicotiana benthamiana plants treated with theobroxide were immediately infiltrated with Pseudomonas syringae pv. tabaci. Exogenous application of theobroxide inhibited development of lesion symptoms, and growth of the bacterial cells was significantly retarded. Semi-quantitative RT-PCRs using the primers of 18 defense-related genes were performed to investigate the molecular mechanisms of resistance. Among the genes, the theobroxide treatment increased the expression of pathogenesis-related protein 1a (PR1a), pathogenesis-related protein 1b (PR1b), glutathione S-transferase (GST), allen oxide cyclase (AOC), and lipoxyganase (LOX). All these data strongly indicate that theobroxide treatment inhibits disease development by faster induction of defense responses, which can be possible by the induction of defense-related genes including PR1a, PR1b, and GST triggered by the elevated jasmonic acid. PMID:25288936

  18. The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

    PubMed

    Limkul, Juthamard; Misaki, Ryo; Kato, Ko; Fujiyama, Kazuhito

    2015-11-01

    Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production.

  19. L-type lectin receptor kinases in Nicotiana benthamiana and tomato and their role in Phytophthora resistance

    PubMed Central

    Wang, Yan; Weide, Rob; Govers, Francine; Bouwmeester, Klaas

    2015-01-01

    Membrane-bound receptors play crucial roles as sentinels of plant immunity against a large variety of invading microbes. One class of receptors known to be involved in self/non-self-surveillance and plant resistance comprises the L-type lectin receptor kinases (LecRKs). Previously, we reported that several Arabidopsis LecRKs play a role in resistance to Phytophthora pathogens. In this study, we determined whether homologues of these LecRKs from the Solanaceous plants Nicotiana benthamiana and tomato (Solanum lycopersicum) play similar roles in defence against Phytophthora. In genome-wide screenings, a total of 38 (Nb)LecRKs were identified in N. benthamiana and 22 (Sl)LecRKs in tomato, each consisting of both a lectin and a kinase domain. Phylogenetic analysis revealed that, in contrast to Arabidopsis, which has a LecRK family comprising nine clades, Solanaceous species have just five of these nine clades (i.e. IV, VI, VII, VIII, and IX), plus four additional clades that lack Arabidopsis homologues. Several of the Solanaceous LecRKs were selected for functional analysis using virus-induced gene silencing. Infection assays with Phytophthora capsici and Phytophthora infestans on LecRK-silenced plants revealed that N. benthamiana and tomato homologues in clade IX play a role in Phytophthora resistance similar to the two Arabidopsis LecRKs in this clade, suggesting conserved functions of clade IX LecRKs across different plant families. This study provides a first insight into the diversity of Solanaceous LecRKs and their role in plant immunity, and shows the potential of LecRKs for Phytophthora resistance breeding. PMID:26248665

  20. Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana.

    PubMed

    Balmuth, Alexi; Rathjen, John P

    2007-09-01

    The Pto gene of tomato (Solanum lycopersicum) confers specific recognition of the unrelated bacterial effector proteins AvrPto and AvrPtoB. Pto resides in a constitutive molecular complex with the nucleotide binding site-leucine rich repeats protein Prf. Prf is absolutely required for specific recognition of both effectors. Here, using stable transgenic lines, we show that expression of Pto from its genomic promoter in susceptible tomatoes was sufficient to complement recognition of Pseudomonas syringae pv. tomato (Pst) bacteria expressing either avrPto or avrPtoB. Pto kinase activity was absolutely required for specific immunity. Expression of the Pto N-myristoylation mutant, pto(G2A), conferred recognition of Pst (avrPtoB), but not Pst (avrPto), although bacterial growth in these lines was intermediate between resistant and susceptible lines. Overexpression of pto(G2A) complemented recognition of avrPto. Transgenic tomato plants overexpressing wild-type Pto exhibited constitutive growth phenotypes, but these were absent in lines overexpressing pto(G2A). Therefore, Pto myristoylation is a quantitative factor for effector recognition in tomato, but is absolutely required for overexpression phenotypes. Native expression of Pto in the heterologous species Nicotiana benthamiana did not confer resistance to P. syringae pv. tabaci (Pta) expressing avrPto or avrPtoB, but recognition of both effectors was complemented by Prf co-expression. Thus, specific resistance conferred solely by Pto in N. benthamiana is an artefact of overexpression. Finally, pto(G2A) did not confer recognition of either avrPto or avrPtoB in N. benthamiana, regardless of the presence of Prf. Thus, co-expression of Prf in N. benthamiana complements many but not all aspects of normal Pto function.

  1. Mitochondrial Alternative Oxidase Maintains Respiration and Preserves Photosynthetic Capacity during Moderate Drought in Nicotiana tabacum1[W

    PubMed Central

    Dahal, Keshav; Wang, Jia; Martyn, Greg D.; Rahimy, Farkhunda; Vanlerberghe, Greg C.

    2014-01-01

    The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum ‘Petit Havana SR1’ wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate drought-stressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO2 and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity. PMID:25204647

  2. Biochemical and Structural Characterization of Benzenoid Carboxyl Methyltransferases Involved in Floral Scent Production in Stephanotis floribunda and Nicotiana suaveolens1

    PubMed Central

    Pott, Marcella B.; Hippauf, Frank; Saschenbrecker, Sandra; Chen, Feng; Ross, Jeannine; Kiefer, Ingrid; Slusarenko, Alan; Noel, Joseph P.; Pichersky, Eran; Effmert, Uta; Piechulla, Birgit

    2004-01-01

    Flower-specific benzenoid carboxyl methyltransferases from Stephanotis floribunda and Nicotiana suaveolens were biochemically and structurally characterized. The floral scents of both these species contain higher levels of methyl benzoate and lower levels of methyl salicylate. The S. floribunda enzyme has a 12-fold lower Km value for salicylic acid (SA) than for benzoic acid (BA), and results of in silico modeling of the active site of the S. floribunda enzyme, based on the crystal structure of Clarkia breweri salicylic acid methyltransferase (SAMT), are consistent with this functional observation. The enzyme was therefore designated SAMT. The internal concentration of BA in S. floribunda flowers is three orders of magnitude higher than the SA concentration, providing a rationale for the observation that these flowers synthesize and emit more methyl benzoate than methyl salicylate. The N. suaveolens enzyme has similar Km values for BA and SA, and the in silico modeling results are again consistent with this in vitro observation. This enzyme was therefore designated BSMT. However, the internal concentration of BA in N. suaveolens petals was also three orders of magnitude higher than the concentration of SA. Both S. floribunda SAMT and N. suaveolens BSMT are able to methylate a range of other benzenoid-related compounds and, in the case of S. floribunda SAMT, also several cinnamic acid derivatives, an observation that is consistent with the larger active site cavity of each of these two enzymes compared to the SAMT from C. breweri, as shown by the models. Broad substrate specificity may indicate recent evolution or an adaptation to changing substrate availability. PMID:15310828

  3. Enhancement of cadmium tolerance and accumulation by introducing Perilla frutescens (L.) Britt var. frutescens genes in Nicotiana tabacum L. plants.

    PubMed

    Wei, Keqiang; Pang, Shengxi; Yang, Junxian; Wei, Zhizhong

    2015-04-01

    The tobacco has the genetic potential to remove toxic metals from the soil. To develop hyperaccumulating tobacco plants, distant hybridization between tobacco (Nicotiana tabacum L.), a high-biomass crop, and Perilla frutescens (L.) Britt var. frutescens, a newfound Cd-hyperaccumulator species, was carried out using a novel method viz. pollination following grafting. Their hybrid nature was preliminarily confirmed by phenotype, isozyme pattern, random amplified polymorphic DNA (RAPD) and metabolites analysis. About 120 putative F2 hybrids derived from the cross-combination [(N. sylvestris Speg. & Comes rootstock + N. tabacum L. var. 78-04 scion) × P. frutescens (L.) Britt var. frutescens] were then subjected to up to 300 μM CdCl2 in hydroponic conditions for 10 days. Results showed five seedlings were more resistant to Cd than female parent and accumulated 314.6 ± 99.9 mg kg(-1) Cd in their aerial biomass, which was 5.7 times greater than that in "78-04" tobacco (47.2 ± 3.56 mg kg(-1)) (P ≤ 0.05). Two of these seedlings exceeded male parent P. frutescens in the Cd concentration of shoots and reached 424 and 396 mg kg(-1), which was 13% and 6% greater for that of perilla (374.2 ± 10.38 mg kg(-1)), respectively. Compared with parents, two other F2 hybrids tended to accumulate more Cd in the root with bioconcentration factor (BCF) 7.05 and 5.17, respectively. Only one hybrid showed lower Cd concentration but transferred Cd more effectively from the root to the shoot than parents and other F2 hybrids, with the maximum translocation factor (TF) value 1.37. These indicated that the introduction of P. frutescens genes could obviously enhance the cadmium tolerance and accumulation of superior individuals.

  4. Expression, subcellular localization, and enzyme activity of a recombinant human extra-cellular superoxide dismutase in tobacco (Nicotiana benthamiana L.).

    PubMed

    Park, Ki Youl; Kim, Eun Yu; Lee, Weontae; Kim, Tae-Yoon; Kim, Woo Taek

    2016-03-01

    Human extracellular superoxide dismutase (hEC-SOD) is an enzyme that scavenges reactive oxygen species (ROS). Because of its antioxidant activity, hEC-SOD has been used as a therapeutic protein to treat skin disease and arthritis in mammalian systems. In this study, codon-optimized hEC-SOD was expressed in tobacco (Nicotiana benthamiana L.) via a plant-based transient protein expression system. Plant expression binary vectors containing full-length hEC-SOD (f-hEC-SOD) and modified hEC-SOD (m-hEC-SOD), in which the signal peptide and heparin-binding domain were deleted, were constructed for the cytosolic-, endoplasmic reticulum (ER)-, and chloroplast-localizations in tobacco leaf mesophyll cells. The results demonstrated that f-hEC-SOD was more efficiently expressed in the cytosolic fractions than in the ER or chloroplasts of tobacco cells. Our data further indicated that differently localized f-hEC-SOD and m-hEC-SOD displayed SOD enzyme activities, suggesting that the hEC-SODs expressed by plants may be functionally active. The f-hEC-SOD was expressed up to 3.8% of the total leaf soluble protein and the expression yield was calculated to be 313.7 μg f-hEC-SOD per g fresh weight of leaf. Overall, our results reveal that it was possible to express catalytically active hEC-SODs by means of a transient plant expression system in tobacco leaf cells. PMID:26611610

  5. L-type lectin receptor kinases in Nicotiana benthamiana and tomato and their role in Phytophthora resistance.

    PubMed

    Wang, Yan; Weide, Rob; Govers, Francine; Bouwmeester, Klaas

    2015-11-01

    Membrane-bound receptors play crucial roles as sentinels of plant immunity against a large variety of invading microbes. One class of receptors known to be involved in self/non-self-surveillance and plant resistance comprises the L-type lectin receptor kinases (LecRKs). Previously, we reported that several Arabidopsis LecRKs play a role in resistance to Phytophthora pathogens. In this study, we determined whether homologues of these LecRKs from the Solanaceous plants Nicotiana benthamiana and tomato (Solanum lycopersicum) play similar roles in defence against Phytophthora. In genome-wide screenings, a total of 38 (Nb)LecRKs were identified in N. benthamiana and 22 (Sl)LecRKs in tomato, each consisting of both a lectin and a kinase domain. Phylogenetic analysis revealed that, in contrast to Arabidopsis, which has a LecRK family comprising nine clades, Solanaceous species have just five of these nine clades (i.e. IV, VI, VII, VIII, and IX), plus four additional clades that lack Arabidopsis homologues. Several of the Solanaceous LecRKs were selected for functional analysis using virus-induced gene silencing. Infection assays with Phytophthora capsici and Phytophthora infestans on LecRK-silenced plants revealed that N. benthamiana and tomato homologues in clade IX play a role in Phytophthora resistance similar to the two Arabidopsis LecRKs in this clade, suggesting conserved functions of clade IX LecRKs across different plant families. This study provides a first insight into the diversity of Solanaceous LecRKs and their role in plant immunity, and shows the potential of LecRKs for Phytophthora resistance breeding.

  6. NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana.

    PubMed

    Saur, Isabel M L; Kadota, Yasuhiro; Sklenar, Jan; Holton, Nicholas J; Smakowska, Elwira; Belkhadir, Youssef; Zipfel, Cyril; Rathjen, John P

    2016-03-22

    Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined.

  7. Chloroplast phosphoglycerate kinase is involved in the targeting of Bamboo mosaic virus to chloroplasts in Nicotiana benthamiana plants.

    PubMed

    Cheng, Shun-Fang; Huang, Ying-Ping; Chen, Li-Hung; Hsu, Yau-Heiu; Tsai, Ching-Hsiu

    2013-12-01

    The Bamboo mosaic virus (BaMV) is a positive-sense, single-stranded RNA virus. Previously, we identified that the chloroplast phosphoglycerate kinase (chl-PGK) from Nicotiana benthamiana is one of the viral RNA binding proteins involved in the BaMV infection cycle. Because chl-PGK is transported to the chloroplast, we hypothesized that chl-PGK might be involved in viral RNA localization in the chloroplasts. To test this hypothesis, we constructed two green fluorescent protein (GFP)-fused mislocalized PGK mutants, the transit peptide deletion mutant (NO TRANSIT PEPTIDE [NOTP]-PGK-GFP) and the nucleus location mutant (nuclear location signal [NLS]-PGK-GFP). Using confocal microscopy, we demonstrated that NOTP-PGK-GFP and NLS-PGK-GFP are localized in the cytoplasm and nucleus, respectively, in N. benthamiana plants. When NOTP-PGK-GFP and NLS-PGK-GFP are transiently expressed, we observed a reduction in BaMV coat protein accumulation to 47% and 27% that of the wild-type PGK-GFP, respectively. To localize viral RNA in infected cells, we employed the interaction of NLS-GFP-MS2 (phage MS2 coat protein) with the modified BaMV RNA containing the MS2 coat protein binding sequence. Using confocal microscopy, we observed that BaMV viral RNA localizes to chloroplasts. Furthermore, elongation factor1a fused with the transit peptide derived from chl-PGK or with a Rubisco small subunit can partially restore BaMV accumulation in NbPGK1-knockdown plants by helping BaMV target chloroplasts.

  8. Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

    PubMed

    Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto

    2014-10-01

    Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

  9. Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

    PubMed

    Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto

    2014-10-01

    Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo. PMID:24964777

  10. Independent, rapid and targeted loss of highly repetitive DNA in natural and synthetic allopolyploids of Nicotiana tabacum.

    PubMed

    Renny-Byfield, Simon; Kovařík, Ales; Chester, Michael; Nichols, Richard A; Macas, Jiri; Novák, Petr; Leitch, Andrew R

    2012-01-01

    Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations. We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor). Using next generation sequencing, a recently developed graph-based repeat identification pipeline, Southern blot and fluorescence in situ hybridisation (FISH) we characterise two highly repetitive DNA sequences (NicCL3 and NicCL7/30). Analysis of two independent high-throughput DNA sequencing datasets indicates NicCL3 forms 1.6-1.9% of the genome in N. tomentosiformis, sequences that occur in multiple, discontinuous tandem arrays scattered over several chromosomes. Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum. Surprisingly elimination of NicCL3 is repeated in some synthetic lines of N. tabacum in their forth generation. The retroelement NicCL7/30, which occurs interspersed with NicCL3, is also under-represented but to a much lesser degree, revealing targeted elimination of the latter. Analysis of paired-end sequencing data indicates the tandem component of NicCL3 has been preferentially removed in natural N. tabacum, increasing the proportion of the dispersed component. This occurs across multiple blocks of discontinuous repeats and based on the distribution of nucleotide similarity among NicCL3 units, was concurrent with rounds of sequence homogenisation. PMID:22606317

  11. Immunodiagnostic Properties of Wucheraria bancrofti SXP-1, a Potential Filarial Diagnostic Candidate Expressed in Tobacco Plant, Nicotiana tabacum.

    PubMed

    Ganapathy, Mathangi; Chakravarthi, M; Charles, S Jason; Harunipriya, P; Jaiganesh, S; Subramonian, N; Kaliraj, P

    2015-08-01

    Transgenic tobacco plants were developed expressing WbSXP-1, a diagnostic antigen isolated from the cDNA library of L3 stage larvae of Wucheraria bancrofti. This antigen produced by recombinant Escherichia coli has been demonstrated by to be successful as potential diagnostic candidate against lymphatic filariasis. A rapid format simple and qualitative flow through immune-filtration diagnostic kit has been developed for the identification of IgG antibodies to the recombinant WbSXP-1 and is being marketed by M/S Span Diagnostics Ltd in India and Africa. Here, we present the results of experiments on the transformation and expression of the same filarial antigen, WbSXP-1, in tobacco plant, Nicotiana tabacum, to produce plant-based diagnostic antigen. It was possible to successfully transform the tobacco plant with WbSXP-1, the integration of the parasite-specific gene in plants was confirmed by PCR amplification and the expression of the filarial protein by Western blotting. The immunoreactivity of the plant-produced WbSXP-1 was assessed based on its reaction with the monoclonal antibodies developed against the E. coli-produced protein. Immunological screening using clinical sera from patients indicates that the plant-produced protein is comparable to E. coli-produced diagnostic antigen. The result demonstrated that plants can be used as suitable expression systems for the production of diagnostic proteins against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. This is the first report of the integration, expression and efficacy of a diagnostic candidate of lymphatic filariasis in plants.Key MessageTransgenic tobacco plants with WbSXP-1, a filarial diagnostic candidate, were developed. The plant-produced protein showed immunoreactivity on par with the E. coli product. PMID:26043851

  12. NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana

    PubMed Central

    Saur, Isabel M. L.; Kadota, Yasuhiro; Sklenar, Jan; Holton, Nicholas J.; Smakowska, Elwira; Belkhadir, Youssef; Zipfel, Cyril; Rathjen, John P.

    2016-01-01

    Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined. PMID:26944079

  13. Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control.

    PubMed

    Gursinsky, Torsten; Pirovano, Walter; Gambino, Giorgio; Friedrich, Susann; Behrens, Sven-Erik; Pantaleo, Vitantonio

    2015-07-01

    The plant ARGONAUTE1 protein (AGO1) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing-based antiviral defense. By genomic and molecular approaches, we here reveal the presence of two homeologs of the AGO1-like gene in Nicotiana benthamiana, NbAGO1-1H and NbAGO1-1L. Both homeologs retain the capacity to transcribe messenger RNAs (mRNAs), which mainly differ in one 18-nucleotide insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located eight nucleotides upstream of the target site of a microRNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGO1-1 homeolog mRNAs at conditions where miR168 is up-regulated, such as during a tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGO1 mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA-induced silencing complexes following the incorporation of small interfering RNAs. Virus-induced gene silencing experiments suggest a specific involvement of the NbAGO1 homeologs in symptom development. The results provide an example of the diversity of microRNA target regions in NbAGO1 homeolog genes, which has important implications for improving resilience measures of the plant during viral infections. PMID:26015446

  14. Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids

    PubMed Central

    2012-01-01

    Background Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units. Methods We examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci. Results and Conclusions In all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations. PMID:23259460

  15. Comparative study on macro- and micro-elements concentration in Nicotiana tabacum and Faba siliquis plants by ICP-MS

    NASA Astrophysics Data System (ADS)

    Balazs, Zoltan; Voica, Cezara; Dehelean, Adriana; Magdas, Dana Alina; Ristoiu, Dumitru

    2015-12-01

    Plants are important components of ecosystems as they transfer elements from abiotic into biotic environments. The concentration of macro and micro-elements in tobacco leaves (Nicotiana tabacum) and bean (Faba siliquis) was analyzed using ICP-MS technique. The results obtained indicated that the mean concentration of Mg, P, K and Ca in tobacco leaves was 0.965, 0.812, 4.412 and 2.694 g.kg-1, respectively, while in bean samples were 0.899, 2.024, 6.725 and 1.387 g.kg-1, respectively. Mn concentration ranged from 156.835 mg.kg-1 to 234.593 mg.kg-1 in tobacco leaves and from 116.174 mg.kg-1 to 440.423 mg.kg-1 in bean samples. The results for Cu and Zn were between 7.262 mg.kg-1 and 105.738 mg.kg-1, 68.549 mg.kg-1 and 113.720 mg.kg-1 (tobacco leaves); and 6.830 mg.kg-1 and 46.034 mg.kg-1, 50.166 mg.kg-1 and 77.242 mg.kg-1 (bean samples), respectively. In analyzed samples, Pb, Cd and As concentrations ranged between <0.001-0.717 mg.kg-1, 0.046 mg.kg-1 -6.218 mg.kg-1, <0.001-0.381 mg.kg-1. The paper discusses the transfer of metal ions (Mn, As, Cd, Cu, Pb and Zn, respectively) from soil to these plants in terms of transfer factors (TF).

  16. NbRABG3f, a member of Rab GTPase, is involved in Bamboo mosaic virus infection in Nicotiana benthamiana.

    PubMed

    Huang, Ying-Ping; Jhuo, Jia-Hua; Tsai, Meng-Shan; Tsai, Ching-Hsiu; Chen, Hong-Chi; Lin, Na-Sheng; Hsu, Yau-Heiu; Cheng, Chi-Ping

    2016-06-01

    The screening of differentially expressed genes in plants after pathogen infection can uncover the potential host factors required for the pathogens. In this study, an up-regulated gene was identified and cloned from Nicotiana benthamiana plants after Bamboo mosaic virus (BaMV) inoculation. The up-regulated gene was identified as a member of the Rab small guanosine triphosphatase (GTPase) family, and was designated as NbRABG3f according to its in silico translated product with high identity to that of RABG3f of tomato. Knocking down the expression of NbRABG3f using a virus-induced gene silencing technique in a protoplast inoculation assay significantly reduced the accumulation of BaMV. A transiently expressed NbRABG3f protein in N. benthamiana plants followed by BaMV inoculation enhanced the accumulation of BaMV to approximately 150%. Mutants that had the catalytic site mutation (NbRABG3f/T22N) or had lost their membrane-targeting capability (NbRABG3f/ΔC3) failed to facilitate the accumulation of BaMV in plants. Because the Rab GTPase is responsible for vesicle trafficking between organelles, a mutant with a fixed guanosine diphosphate form was used to identify the donor compartment. The use of green fluorescent protein (GFP) fusion revealed that GFP-NbRABG3f/T22N clearly co-localized with the Golgi marker. In conclusion, BaMV may use NbRABG3f to form vesicles derived from the Golgi membrane for intracellular trafficking to deliver unidentified factors to its replication site; thus, both GTPase activity and membrane-targeting ability are crucial for BaMV accumulation at the cell level.

  17. Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene induced by sulphur starvation.

    PubMed

    Wawrzyńska, Anna; Lewandowska, Małgorzata; Sirko, Agnieszka

    2010-03-01

    Sulphur deficiency severely affects plant growth and their agricultural productivity leading to diverse changes in development and metabolisms. Molecular mechanisms regulating gene expression under low sulphur conditions remain largely unknown. AtSLIM1, a member of the EIN3-like (EIL) family was reported to be a central transcriptional regulator of the plant sulphur response, however, no direct interaction of this protein with any sulphur-responsive promoters was demonstrated. The focus of this study was on the analysis of a promoter region of UP9C, a tobacco gene strongly induced by sulphur limitation. Cloning and subsequent examination of this promoter resulted in the identification of a 20-nt sequence (UPE-box), also present in the promoters of several Arabidopsis genes, including three out of four homologues of UP9C. The UPE-box, consisting of two parallel tebs sequences (TEIL binding site), proved to be necessary to bind the transcription factors belonging to the EIL family and of a 5-nt conserved sequence at the 3'-end. The yeast one-hybrid analysis resulted in the identification of one transcription factor (NtEIL2) capable of binding to the UPE-box. The interactions of NtEIL2, and its homologue from Arabidopsis, AtSLIM1, with DNA were affected by mutations within the UPE-box. Transient expression assays in Nicotiana benthamiana have further shown that both factors, NtEIL2 and AtSLIM1, activate the UP9C promoter. Interestingly, activation by NtEIL2, but not by AtSLIM1, was dependent on the sulphur-deficiency of the plants.

  18. Immunodiagnostic Properties of Wucheraria bancrofti SXP-1, a Potential Filarial Diagnostic Candidate Expressed in Tobacco Plant, Nicotiana tabacum.

    PubMed

    Ganapathy, Mathangi; Chakravarthi, M; Charles, S Jason; Harunipriya, P; Jaiganesh, S; Subramonian, N; Kaliraj, P

    2015-08-01

    Transgenic tobacco plants were developed expressing WbSXP-1, a diagnostic antigen isolated from the cDNA library of L3 stage larvae of Wucheraria bancrofti. This antigen produced by recombinant Escherichia coli has been demonstrated by to be successful as potential diagnostic candidate against lymphatic filariasis. A rapid format simple and qualitative flow through immune-filtration diagnostic kit has been developed for the identification of IgG antibodies to the recombinant WbSXP-1 and is being marketed by M/S Span Diagnostics Ltd in India and Africa. Here, we present the results of experiments on the transformation and expression of the same filarial antigen, WbSXP-1, in tobacco plant, Nicotiana tabacum, to produce plant-based diagnostic antigen. It was possible to successfully transform the tobacco plant with WbSXP-1, the integration of the parasite-specific gene in plants was confirmed by PCR amplification and the expression of the filarial protein by Western blotting. The immunoreactivity of the plant-produced WbSXP-1 was assessed based on its reaction with the monoclonal antibodies developed against the E. coli-produced protein. Immunological screening using clinical sera from patients indicates that the plant-produced protein is comparable to E. coli-produced diagnostic antigen. The result demonstrated that plants can be used as suitable expression systems for the production of diagnostic proteins against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. This is the first report of the integration, expression and efficacy of a diagnostic candidate of lymphatic filariasis in plants.Key MessageTransgenic tobacco plants with WbSXP-1, a filarial diagnostic candidate, were developed. The plant-produced protein showed immunoreactivity on par with the E. coli product.

  19. L-type lectin receptor kinases in Nicotiana benthamiana and tomato and their role in Phytophthora resistance.

    PubMed

    Wang, Yan; Weide, Rob; Govers, Francine; Bouwmeester, Klaas

    2015-11-01

    Membrane-bound receptors play crucial roles as sentinels of plant immunity against a large variety of invading microbes. One class of receptors known to be involved in self/non-self-surveillance and plant resistance comprises the L-type lectin receptor kinases (LecRKs). Previously, we reported that several Arabidopsis LecRKs play a role in resistance to Phytophthora pathogens. In this study, we determined whether homologues of these LecRKs from the Solanaceous plants Nicotiana benthamiana and tomato (Solanum lycopersicum) play similar roles in defence against Phytophthora. In genome-wide screenings, a total of 38 (Nb)LecRKs were identified in N. benthamiana and 22 (Sl)LecRKs in tomato, each consisting of both a lectin and a kinase domain. Phylogenetic analysis revealed that, in contrast to Arabidopsis, which has a LecRK family comprising nine clades, Solanaceous species have just five of these nine clades (i.e. IV, VI, VII, VIII, and IX), plus four additional clades that lack Arabidopsis homologues. Several of the Solanaceous LecRKs were selected for functional analysis using virus-induced gene silencing. Infection assays with Phytophthora capsici and Phytophthora infestans on LecRK-silenced plants revealed that N. benthamiana and tomato homologues in clade IX play a role in Phytophthora resistance similar to the two Arabidopsis LecRKs in this clade, suggesting conserved functions of clade IX LecRKs across different plant families. This study provides a first insight into the diversity of Solanaceous LecRKs and their role in plant immunity, and shows the potential of LecRKs for Phytophthora resistance breeding. PMID:26248665

  20. Induction of UDP-glucose:salicylic acid glucosyltransferase activity in tobacco mosaic virus-inoculated tobacco (Nicotiana tabacum) leaves

    SciTech Connect

    Enyedi, A.J.; Raskin, I. )

    1993-04-01

    Salicylic acid (SA) is a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco masaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. The SA increase in the inoculated leaf coincided with the appearance of a [beta]-glucosidase-hydrolyzable SA conjugate identified as [beta]-O-D-glucosylsalicylic acid (GSA). SA and GSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) ([beta]-GTase) capable of converting SA to GSA. Healthy tissues had constitutive [beta]-GTase activity of 0.076 milliunits g[sup [minus]1] fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated [beta]-GTase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. The effect of TMV inoculation on the [beta]-GTase and GSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMV-inoculated leaves (2.7--27.0 [mu]g g[sup [minus]1] fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of [beta]GTase by SA and prevented the formation of GSA. Of 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced [beta]-GTase activity. 21 refs., 5 figs.

  1. Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control1[OPEN

    PubMed Central

    Gursinsky, Torsten; Gambino, Giorgio; Friedrich, Susann; Behrens, Sven-Erik

    2015-01-01

    The plant ARGONAUTE1 protein (AGO1) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing-based antiviral defense. By genomic and molecular approaches, we here reveal the presence of two homeologs of the AGO1-like gene in Nicotiana benthamiana, NbAGO1-1H and NbAGO1-1L. Both homeologs retain the capacity to transcribe messenger RNAs (mRNAs), which mainly differ in one 18-nucleotide insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located eight nucleotides upstream of the target site of a microRNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGO1-1 homeolog mRNAs at conditions where miR168 is up-regulated, such as during a tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGO1 mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA-induced silencing complexes following the incorporation of small interfering RNAs. Virus-induced gene silencing experiments suggest a specific involvement of the NbAGO1 homeologs in symptom development. The results provide an example of the diversity of microRNA target regions in NbAGO1 homeolog genes, which has important implications for improving resilience measures of the plant during viral infections. PMID:26015446

  2. Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses.

    PubMed

    Wang, Ken-Der; Empleo, Roman; Nguyen, Tan Tri V; Moffett, Peter; Sacco, Melanie Ann

    2015-06-01

    Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0(T) (u) ), PLRV (P0(PL) ) and Cucurbit aphid-borne yellows virus (P0(CA) ) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0(PL) only. Genetic analysis showed that recognition of P0(T) (u) by a resistance gene designated RPO1 (Resistance to POleroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0(T) (u) mutants with substitutions in the F-box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F-box protein motif could indicate detection of P0-induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance.

  3. NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana.

    PubMed

    Saur, Isabel M L; Kadota, Yasuhiro; Sklenar, Jan; Holton, Nicholas J; Smakowska, Elwira; Belkhadir, Youssef; Zipfel, Cyril; Rathjen, John P

    2016-03-22

    Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined. PMID:26944079

  4. Nep1-like protein from Moniliophthora perniciosa induces a rapid proteome and metabolome reprogramming in cells of Nicotiana benthamiana.

    PubMed

    Villela-Dias, Cristiano; Camillo, Luciana R; de Oliveira, Guilherme A P; Sena, Jamilly A L; Santiago, André S; de Sousa, Sanderson T P; Mendes, Juliano S; Pirovani, Carlos P; Alvim, Fátima C; Costa, Marcio G C

    2014-01-01

    NEP1 (necrosis- and ethylene-inducing peptide 1)-like proteins (NLPs) have been identified in a variety of taxonomically unrelated plant pathogens and share a common characteristic of inducing responses of plant defense and cell death in dicotyledonous plants. Even though some aspects of NLP action have been well characterized, nothing is known about the global range of modifications in proteome and metabolome of NLP-treated plant cells. Here, using both proteomic and metabolomic approaches we were able to identify the global molecular and biochemical changes in cells of Nicotiana benthamiana elicited by short-term treatment with MpNEP2, a NLP of Moniliophthora perniciosa, the basidiomycete responsible for the witches' broom disease on cocoa (Theobroma cacao L.). Approximately 100 protein spots were collected from 2-DE gels in each proteome, with one-third showing more than twofold differences in the expression values. Fifty-three such proteins were identified by mass spectrometry (MS)/MS and mapped into specific metabolic pathways and cellular processes. Most MpNEP2 upregulated proteins are involved in nucleotide-binding function and oxidoreductase activity, whereas the downregulated proteins are mostly involved in glycolysis, response to stress and protein folding. Thirty metabolites were detected by gas spectrometry (GC)/MS and semi-quantified, of which eleven showed significant differences between the treatments, including proline, alanine, myo-inositol, ethylene, threonine and hydroxylamine. The global changes described affect the reduction-oxidation reactions, ATP biosynthesis and key signaling molecules as calcium and hydrogen peroxide. These findings will help creating a broader understanding of NLP-mediated cell death signaling in plants. PMID:23586401

  5. Induction of a small heat shock protein and its functional roles in Nicotiana plants in the defense response against Ralstonia solanacearum.

    PubMed

    Maimbo, Milimo; Ohnishi, Kouhei; Hikichi, Yasufumi; Yoshioka, Hirofumi; Kiba, Akinori

    2007-12-01

    In tobacco (Nicotiana tabacum), Ralstonia solanacearum OE1-1 (RsOE1-1) is pathogenic, whereas R. solanacearum 8107 (Rs8107) is nonpathogenic and induces the hypersensitive response (HR). To elucidate the molecular mechanisms of plant-R. solanacearum interactions, we used differential display to isolate a cDNA fragment, A6, regulated in tobacco by inoculation with RsOE1-1. The deduced amino acid sequence predicted from full-length A6-cDNA showed similarity to small heat shock proteins from Arabidopsis (Arabidopsis thaliana; hypothetical protein), Medicago truncatula, and Cucumis melo; we therefore designated A6 to correspond to Ntshsp17 (for tobacco small heat shock protein 17). Recombinant Ntshsp17 overproduced in Escherichia coli exhibited molecular chaperone function. Expression of Ntshsp17 was increased in tobacco leaves inoculated with both RsOE1-1 and Rs8107. Expression was induced by heat treatment and by treatment with aminocyclopropane carboxylic acid, hydrogen peroxide, methyl jasmonate, and salicylic acid. Ntshsp17 expression was induced by inoculation with a HR and pathogenicity gene mutant of Rs8107 that does not induce the HR, but not by Agrobacterium-mediated transient expression of INF1, an HR elicitor. In Nbshsp17-silenced plants (an Ntshsp17 ortholog in Nicotiana benthamiana), expression of ETHYLENE-RESPONSE ELEMENT-BINDING PROTEIN, PATHOGENESIS-RELATED1a (PR1a), and PR4 genes was compromised, but expression of ELONGATION FACTOR1alpha was scarcely affected. Appearance of the HR was not affected in the silenced plants. In the silenced plants, growth of Rs8107 was accelerated. Bacterial growth and wilt symptoms elicited by RsOE1-1 were also accelerated in the silenced plants. These results indicate that this small heat shock protein might have a role in HR-independent defenses in Nicotiana plants. PMID:17965181

  6. Reduced abundance of the CYP6CY3-targeting let-7 and miR-100 miRNAs accounts for host adaptation of Myzus persicae nicotianae.

    PubMed

    Peng, Tianfei; Pan, Yiou; Gao, Xiwu; Xi, Jinghui; Zhang, Lei; Ma, Kangsheng; Wu, Yongqiang; Zhang, Juhong; Shang, Qingli

    2016-08-01

    Nicotine is one of the most abundant and toxic secondary plant metabolites in nature and is defined by high toxicity to plant-feeding insects. Studies suggest that increased expression of cytochrome P450 (CYP6CY3) and the homologous CYP6CY4 genes in Myzus persicae nicotianae is correlated with tolerance to nicotine. Indeed, through expression analyses of the CYP6CY3 and CYP6CY4 genes of different M. persicae subspecies, we determined that the mRNA levels of these two genes were much higher in M. persicae nicotianae than in M. persicae sensu stricto. We hypothesized that the expression of these two genes is subject to post-transcriptional regulation. To investigate the underlying mechanism, the miRNA profile of M. persicae nicotianae was sequenced, and twenty-two miRNAs were predicted to target CYP6CY3. Validation of these miRNAs identified two miRNAs, let-7 and miR-100, whose abundance was highly inversely correlated with the abundance of the CYP6CY3 gene. This result implies that the let-7 and miR-100 miRNAs play a major role in the post-transcriptional regulation of the CYP6CY3 gene. Modulation of the abundance of let-7 and miR-100 through the addition of inhibitors/mimics of let-7 or miR-100 to artificial diet significantly altered the tolerance of M. persicae nicotianae to nicotine, further confirming the regulatory role of these two miRNAs. Interestingly, after decreasing the transcript levels of CYP6CY3 by modulating regulatory miRNAs, the transcript levels of the homologous isozyme CYP6CY4 were significantly elevated, suggesting a compensatory mechanism between the CYP6CY3 gene and its homologous CYP6CY4 gene. Our findings provide insight into the molecular drivers of insect host shifts and reveal an important source of genetic variation for adaptive evolution in insect species. PMID:27318250

  7. Synthesis of viral DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with cassava latent virus (CLV); evidence for the independent replication of one component of the CLV genome.

    PubMed Central

    Townsend, R; Watts, J; Stanley, J

    1986-01-01

    Totipotent leaf mesophyll protoplasts of Nicotiana plumbaginifolia, Viviani were inoculated with cassava latent virus (CLV) or with full length copies of CLV genomic DNAs 1 and 2 excised from replicative forms of M13 clones. Virus specific DNAs began to appear 48-72h after inoculation with virus or cloned DNAs, coincident with the onset of host cell division. Infected cells accumulated supercoiled forms of DNAs 1 and 2 as well as progeny single-stranded (ss) virion (+) sense DNAs representing each component of the genome. Both supercoiled and ss molecules were synthesised by cells inoculated with cloned DNA 1 alone but DNA 2 failed to replicate independently. Images PMID:3951986

  8. Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid-mediated systemic virus resistance in Nicotiana benthamiana.

    PubMed

    Deng, Xing-Guang; Zhu, Tong; Zou, Li-Juan; Han, Xue-Ying; Zhou, Xue; Xi, De-Hui; Zhang, Da-Wei; Lin, Hong-Hui

    2016-02-01

    Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H2O2 and NO. Scavenging of H2O2 or NO in upper leaves blocked BR-induced systemic virus resistance. BR-induced systemic H2O2 accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite-dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR-triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR-activated H2O2 is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H2O2 generation blocked BR-induced systemic NO production, but BR-induced H2O2 production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR-induced systemic virus defense in NbRBOHB-silenced plants, but H2O2 did not reverse the effect of NbNR silencing on BR-induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR-mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR-induced H2O2 and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR-mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)-dependent H2O2 production and subsequent systemic NR-dependent NO generation. PMID:26749255

  9. Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana.

    PubMed

    Zhang, Donghui; Jasieniecka-Gazarkiewicz, Katarzyna; Wan, Xia; Luo, Ling; Zhang, Yinbo; Banas, Antoni; Jiang, Mulan; Gong, Yangmin

    2015-01-01

    In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid

  10. Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana

    PubMed Central

    Wan, Xia; Luo, Ling; Zhang, Yinbo; Banas, Antoni; Jiang, Mulan; Gong, Yangmin

    2015-01-01

    In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid

  11. A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update

    PubMed Central

    Ambrós, Silvia; Ruiz-Ruiz, Susana; Peña, Leandro; Moreno, Pedro

    2013-01-01

    In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system. PMID

  12. The role of mesophyll conductance during water stress and recovery in tobacco (Nicotiana sylvestris): acclimation or limitation?

    PubMed

    Galle, Alexander; Florez-Sarasa, Igor; Tomas, Magdalena; Pou, Alicia; Medrano, Hipolito; Ribas-Carbo, Miquel; Flexas, Jaume

    2009-01-01

    While the responses of photosynthesis to water stress have been widely studied, acclimation to sustained water stress and recovery after re-watering is poorly understood. In particular, the factors limiting photosynthesis under these conditions, and their possible interactions with other environmental conditions, are unknown. To assess these issues, changes of photosynthetic CO(2) assimilation (A(N)) and its underlying limitations were followed during prolonged water stress and subsequent re-watering in tobacco (Nicotiana sylvestris) plants growing under three different climatic conditions: outdoors in summer, outdoors in spring, and indoors in a growth chamber. In particular, the regulation of stomatal conductance (g(s)), mesophyll conductance to CO(2) (g(m)), leaf photochemistry (chlorophyll fluorescence), and biochemistry (V(c,max)) were assessed. Leaf gas exchange and chlorophyll fluorescence data revealed that water stress induced a similar degree of stomatal closure and decreased A(N) under all three conditions, while V(c,max) was unaffected. However, the behaviour of g(m) differed depending on the climatic conditions. In outdoor plants, g(m) strongly declined with water stress, but it recovered rapidly (1-2 d) after re-watering in spring while it remained low many days after re-watering in summer. In indoor plants, g(m) initially declined with water stress, but then recovered to control values during the acclimation period. These differences were reflected in different velocities of recovery of A(N) after re-watering, being the slowest in outdoor summer plants and the fastest in indoor plants. It is suggested that these differences among the experiments are related to the prevailing climatic conditions, i.e. to the fact that stress factors other than water stress have been superimposed (e.g. excessive light and elevated temperature). In conclusion, besides g(s), g(m) contributes greatly to the limitation of photosynthesis during water stress and during

  13. Acidic α-galactosidase is the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum)

    PubMed Central

    Zha, Hong-Guang; Flowers, V. Lynn; Yang, Min; Chen, Ling-Yang; Sun, Hang

    2012-01-01

    Background and Aims To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems. Methods Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied. Key Results The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5·33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by d-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development. Conclusions α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco

  14. Uridine Diphosphate Glucose Metabolism and Callose Synthesis in Cultured Pollen Tubes of Nicotiana alata Link et Otto.

    PubMed Central

    Schlupmann, H.; Bacic, A.; Read, S. M.

    1994-01-01

    Membrane preparations from cultured pollen tubes of Nicotiana alata Link et Otto contain a Ca2+ -independent (1-3)-[beta]-D-glucan (callose) synthase activity that has a low affinity for UDP-glucose, even when activated by treatment with trypsin (H. Schlupmann, A. Basic, S.M. Read [1993] Planta 191: 470-481). Therefore, we investigated whether UDP-glucose was a likely substrate for callose synthesis in actively growing pollen tubes. Deposition of (1-3)-[beta]-glucan occurred at a constant rate, 1.4 to 1.7 nmol glucose min-1, in tubes from 1 mg of pollen from 3 h after germination; however, the rate of incorporation of radioactivity from exogenous [14C]-sucrose into wall polymers was not constant, but increased until at least 8 h after germination, probably due to decreasing use of internal reserves. UDP-glucose was a prominent ultraviolet-absorbing metabolite in pollen-tube extracts, with 1.6 nmol present in tubes from 1 mg of pollen, giving a calculated cytoplasmic concentration of approximately 3.5 mM. Radioactivity from [14C]-sucrose was rapidly incorporated into sugar monophosphates and UDP-glucose by the growing tubes, consistent with a turnover time for UDP-glucose of less than 1 min; the specific radioactivity of extracted UDP-[14C]glucose was equal to that calculated from the rate of incorporation of [14C]sucrose into wall glucans. Large amounts of less metabolically active neutral sugars were also present. The rate of synthesis of (1-3)-[beta]-glucan by nontrypsin-treated pollen-tube membrane preparations incubated with 3.5 mM UDP-glucose and a [beta]-glucoside activator was slightly greater than the rate of deposition of (1-3)-[beta]-glucan by intact pollen tubes. These data are used to assess the physiological significance of proteolytic activation of pollen-tube callose synthase. PMID:12232233

  15. Effect of UV irradiation, toluidine blue, and environment on maternal haploid frequencies from the cross between Nicotiana tabacum and N. africana

    SciTech Connect

    Chimoyo, H.M.

    1988-01-01

    Treating Nicotiana africana Merxm. pollen with three levels UV radiation prior to pollinating four cultivars of flue-cured tobacco (Coker 176, NC95, McN944 and PD4), Nicotiana tabacum produced 1,953 viable seedlings from an estimated total of 170,248 seeds, of which 1,667 were haploid and 286 were hybrids. Drenching N. tabacum flowers with toluidine blue 18 hours after pollination with normal N. africana pollen, yielded 511 viable seedlings from 70,613 seeds, of which 346 were haploid and 165 hybrids. Untreated pollen gave 548 viable seedlings from 56,291 seeds, comprising 341 haploids and 208 hybrids. Contrary to results from a previous histological study, in vivo pollen tube growth rate appears to be similar irrespective of pollen source or treatment, and fertilization seems to occur at about the same time as in the selfed control. From an estimated total of 803,854 seeds sown, 3,014 viable seedlings were obtained. Coker 176 gave significantly higher yields of haploids than the other three cultivars. Field grown plants produced more haploids than greenhouse grown plants. Further evidence was obtained to support selective chromosomal elimination as the mechanism governing the development of maternal haploids from this interspecific cross.

  16. The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana.

    PubMed

    Shibata, Yusuke; Ojika, Makoto; Sugiyama, Akifumi; Yazaki, Kazufumi; Jones, David A; Kawakita, Kazuhito; Takemoto, Daigo

    2016-05-01

    The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans.

  17. Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

    PubMed

    Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

    2016-08-01

    Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG.

  18. Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

    PubMed

    Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

    2016-08-01

    Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG. PMID:27125327

  19. Novel type of adenylyl cyclase participates in tabtoxinine-β-lactam-induced cell death and occurrence of wildfire disease in Nicotiana benthamiana.

    PubMed

    Ito, Makoto; Takahashi, Hirotaka; Sawasaki, Tatsuya; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2014-01-01

    Tabtoxinine-β-lactam (TβL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TβL causes the plant cell death by the inhibiting glutamine synthetase, which leads to an abnormal accumulation of ammonium ions. To better understand the molecular mechanisms involved in TβL-induced cell death and necrotic wildfire lesions, we focused on adenylyl cyclase in Nicotiana benthamiana. We isolated the gene designated as NbAC (Nicotiana benthamiana adenylyl cyclase). Recombinant NbAC protein showed adenylyl cyclase activity in vitro. TβL-induced necrotic lesions were significantly suppressed in NbAC-silenced leaves compared with control plant leaves. However, the amount of ammonium ions was scarcely affected by NbAC-silencing. Furthermore, the silencing of NbAC also suppressed l-methionine sulfoximine-induced cell death without any changes in the amount of ammonium accumulated. When inoculated directly with P. syringae pv tabaci, NbAC-silenced plants showed reduced symptoms. These results suggest that NbAC might play an essential role in intracellular signal transduction during TβL-induced cell death and necrotic wildfire disease development.

  20. Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation 1

    PubMed Central

    Campell, Bruce R.; Su, Ling-Yuan; Pengelly, William L.

    1989-01-01

    Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N. glutinosa tumor cells was inhibited by addition of α-naphthaleneacetic acid to the growth medium, and A6- and A66-transformed cells showed similar dose responses to this auxin. On the other hand, A6-transformed cells contained much higher levels of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) than A66-transformed cells. Differences in IAA and ACC levels in N. glutinosa tumor lines were consistent with the expected activity of the tms locus and were quantitatively similar to results obtained previously with A6- and A66-transformed cells of Nicotiana tabacum, which does not compensate for mutated tms genes. Thus, compensation for mutated tms genes in N. glutinosa did not result from increased auxin accumulation and did not appear to be related to the capacity of this host for auxin biosynthesis. PMID:16666706

  1. Ovipositional response of tobacco budworm moths (Lepidoptera: Noctuidae) to cuticular labdanes and sucrose esters from the green leaves ofNicotiana glutinosa L. (Solanaceae).

    PubMed

    Jackson, D M; Severson, R F; Sisson, V A; Stephenson, M G

    1991-12-01

    Field plots of three accessions ofNicotiana glutinosa L. (Nicotiana species accessions 24, 24A, and 24B) at Oxford, North Carolina and Tifton, Georgia were heavily damaged by natural populations of tobacco budworms,Heliothis virescens (F.), during 1985-1989. Experiments in outdoor screen cages demonstrated that all accessions ofN. glutinosa were as prone to oviposition byH. virescens moths as was NC 2326, a commercial cultivar of flue-cured tobacco,N. tabacum L. However, in greenhouse experiments, tobacco budworm larvae did not survive or grow as well when placed on plants ofN. glutinosa as they did when placed on plants of NC 2326. Four labdane diterpenes (manool, 2-hydroxymanool, a mixture of sclareols, and labda-13-ene-8α,15-diol [labdenediol]) and two sucrose ester fractions (2,3,4-tri-O-acyl-3'-O-acetyl-sucrose [G-SE-I] and 2,3,4,-tri-O-acyl-sucrose [G-SE-II]) were isolated from green leaves of the three accessions ofN. glutinosa. These components were bioassayed for their effects on the ovipositional behavior of tobacco budworm moths using small screen cages in a greenhouse at Oxford, North Carolina. Labdenediol, manool, and both sucrose ester fractions stimulated tobacco budworm moths to oviposit on a tobacco budworm-resistant Tobacco Introduction, TI 1112 (PI 124166), when these materials were sprayed onto a leaf. PMID:24258642

  2. Differences in intensity and specificity of hypersensitive response induction in Nicotiana spp. by INF1, INF2A, and INF2B of Phytophthora infestans.

    PubMed

    Huitema, Edgar; Vleeshouwers, Vivianne G A A; Cakir, Cahit; Kamoun, Sophien; Govers, Francine

    2005-03-01

    Elicitins form a family of structurally related proteins that induce the hypersensitive response (HR) in plants, particularly Nicotiana spp. The elicitin family is composed of several classes. Most species of the plant-pathogenic oomycete genus Phytophthora produce the well-characterized 10-kDa canonical elicitins (class I), such as INF1 of the potato and tomato pathogen Phytophthora infestans. Two genes, inf2A and inf2B, encoding a distinct class (class III) of elicitin-like proteins, also occur in P. infestans. Unlike secreted class I elicitins, class III elicitins are thought to be cell-surface-anchored polypeptides. Molecular characterization of the inf2 genes indicated that they are widespread in Phytophthora spp. and occur as a small gene family. In addition, Southern blot and Northern blot hybridizations using gene-specific probes showed that inf2A and inf2B genes and transcripts can be detected in 17 different P. infestans isolates. Functional secreted expression in plant cells of the elicitin domain of the infl and inf2 genes was conducted using a binary Potato virus X (PVX) vector (agroinfection) and Agrobacterium tumefaciens transient transformation assays (agroinfiltration), and resulted in HR-like necrotic symptoms and induction of defense response genes in tobacco. However, comparative analyses of elicitor activity of INF1, INF2A, and INF2B revealed significant differences in intensity, specificity, and consistency of HR induction. Whereas INF1 induced the HR in Nicotiana benthamiana, INF2A induced weak symptoms and INF2B induced no symptoms on this plant. Nonetheless, similar to INF1, HR induction by INF2A in N. benthamiana required the ubiquitin ligase-associated protein SGT1. Overall, these results suggest that variation in the resistance of Nicotiana spp. to P. infestans is shadowed by variation in the response to INF elicitins. The ability of tobacco, but not N. benthamiana, to respond to INF2B could explain differences in resistance to P

  3. Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata.

    PubMed

    Dracatos, Peter M; van der Weerden, Nicole L; Carroll, Kate T; Johnson, Elizabeth D; Plummer, Kim M; Anderson, Marilyn A

    2014-01-01

    Defensins are a large family of small, cysteine-rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C-terminal pro-peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt-thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC50 ) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5-10 times less effective at killing necrotrophs, but only two-fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat

  4. Spectral reflectance, chlorophyll fluorescence and virological investigations of tobacco plants (Nicotiana tabacum L.) infected with Tobacco mosaic virus (TMV)

    NASA Astrophysics Data System (ADS)

    Krezhova, Dora; Hristova, Dimitrina; Iliev, Ilko; Yanev, Tony

    Application of multispectral remote sensing techniques to plant condition monitoring has been adopted for various purposes. Remote sensing is a reliable tool for detecting signs of vege-tation stress and diseases. Spectral reflectance and chlorophyll fluorescence are functions of tissue optical properties and biological status of the plants, and illumination conditions. The mean reflectance spectrum depends on the relative composition of all the pigments in the leaf including chlorophylls, carotenoids etc. Chlorophyll fluorescence results from the primary re-actions of photosynthesis and during the last decade it finds widening application as a means for revelation of stress and diseases. The changes in chlorophyll function take place before the alteration in chlorophyll content to occur so that changes in the fluorescence signal arise before any visible signs are apparent. The aim of our investigations was to study the development and spreading out of a viral infection on the leaves of two cultivars tobacco plants (Nicotiana tabacum L.) infected with Tobacco mosaic virus (TMV). We applied two remote sensing tech-niques (spectral reflectance and chlorophyll fluorescence measurements) for evaluation of the changes in the optical properties of the plants in accordance to their physiological status. The serological analyses via the Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) were made with appropriate kits (Leowe, Germany) for quantitative assessment of the concentration of viruses in the plants. The tobacco plants were grown in green house under controlled conditions. The first cultivar Nevrocop 1146 is known as resistive to the TMV, i.e. it shows hypersensitive response. The second cultivar named Krumovgrad is normally sen-sitive to the TMV. At growth stage 4-6 expanded leaf, up to one leaf from 20 plants for each cultivar were inoculated with TMV. The leaves opposite to the infected ones formed the group of control (untreated) leaves. The

  5. A new virus-induced gene silencing vector based on Euphorbia mosaic virus-Yucatan peninsula for NPR1 silencing in Nicotiana benthamiana and Capsicum annuum var. Anaheim.

    PubMed

    Villanueva-Alonzo, Hernan J; Us-Camas, Rosa Y; López-Ochoa, Luisa A; Robertson, Dominique; Guerra-Peraza, Orlene; Minero-García, Yereni; Moreno-Valenzuela, Oscar A

    2013-05-01

    Virus-induced gene silencing is based on the sequence-specific degradation of RNA. Here, a gene silencing vector derived from EuMV-YP, named pEuMV-YP:ΔAV1, was used to silence ChlI and NPR1 genes in Nicotiana benthamiana. The silencing of the ChlI transcripts was efficient in the stems, petioles and leaves as reflected in tissue bleaching and reduced transcript levels. The silencing was stable, reaching the flowers and fruits, and was observed throughout the life cycle of the plants. Additionally, the silencing of the NPR1 gene was efficient in both N. benthamiana and Capsicum annuum. After silencing, the plants' viral symptoms increased to levels similar to those seen in wild-type plants. These results suggest that NPR1 plays a role in the compatible interactions of EuMV-YP N. benthamiana and EuMV-C. annum var. anaheim.

  6. Isolation and characterization of NgRLK1, a receptor-like kinase of Nicotiana glutinosa that interacts with the elicitin of Phytophthora capsici.

    PubMed

    Kim, Yeong-Tae; Oh, Jonghee; Kim, Kyung-Hwan; Uhm, Jae-Youl; Lee, Byoung-Moo

    2010-02-01

    Elicitins, extracellular proteins from Phytophthora fungi, elicit a hypersensitivity response (HR), including systemic acquired resistance, in some plants. The elicitin capsicein (approximately 10 kDa) was purified by FPLC from culture filtrates of P. capsici. Purified native and recombinant capsicein induced a hypersensitive response in leaves of the non-host plants Nicotiana glutinosa and Brassica rapa subsp. pekinensis. To search for candidate capsicein-interacting proteins from N. glutinosa, a yeast two-hybrid assay was used. We identified a protein interactor that is homologous to a serine/threonine kinase of the plant receptor-like kinase (RLK) group and designated it NgRLK1. The ORF of NgRLK1 encodes a polypeptide of 832 amino acids (93,490 Da). A conserved domain analysis revealed that NgRLK1 has structural features typical of a plant RLK. NgRLK1 was autophosphorylated, with higher activity in the presence of Mn2+ than Mg2+.

  7. The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

    PubMed Central

    Resmi, Thulasi Raveendrannair; Hohn, Thomas; Hohn, Barbara; Veluthambi, Karuppannan

    2015-01-01

    Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases. PMID:26008704

  8. The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants.

    PubMed

    Resmi, Thulasi Raveendrannair; Hohn, Thomas; Hohn, Barbara; Veluthambi, Karuppannan

    2015-05-01

    Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases. PMID:26008704

  9. Uptake of NO, NO 2 and O 3 by sunflower ( Helianthus annuus L.) and tobacco plants ( Nicotiana tabacum L.): dependence on stomatal conductivity

    NASA Astrophysics Data System (ADS)

    Neubert, A.; Kley, D.; Wildt, J.; Segschneider, H. J.; Förstel, H.

    The uptake of NO, NO 2 and O 3 by sunflowers ( Helianthus annuus L. var. giganteus) and tobacco plants ( Nicotiana tabacum L. var. Bel W3), using concentrations representative for moderately polluted air, has been determined by gas exchange experiments. Conductivities for these trace gases were measured at different light fluxes ranging from 820 μEm -2s -1 to darkness. The conductivities to water vapor and the trace gases are highly correlated. It is concluded that the uptake of NO, NO 2 and O 3 by sunflowers and tobacco plants is linearly dependent on stomatal opening. While the uptake of NO is limited by the mesophyll resistance, the uptake of NO 2 is only by diffusion through the stomata. Loss processes by deposition to the leaf surfaces are more pronounced for O 3 than for NO and NO 2.

  10. Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells

    SciTech Connect

    Kleinow, Tatjana; Tanwir, Fariha; Kocher, Cornelia; Krenz, Bjoern; Wege, Christina; Jeske, Holger

    2009-09-01

    The geminivirus Abutilon mosaic virus (AbMV) encodes two proteins which are essential for viral spread within plants. The nuclear shuttle protein (NSP) transfers viral DNA between the nucleus and cytoplasm, whereas the movement protein (MP) facilitates transport between cells through plasmodesmata and long-distance via phloem. An inducible overexpression system for epitope-tagged NSP and MP in plants yielded unprecedented amounts of both proteins. Western blots revealed extensive posttranslational modification and truncation for MP, but not for NSP. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when epitope-tagged NSP and MP were simultaneously expressed in leaves locally infected with an AbMV DNA A in which the coat protein gene was replaced by a green fluorescent protein encoding gene. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. MP appeared at the cell periphery, probably the plasma membrane, and plasmodesmata.

  11. Molecular chaperons and co-chaperons, Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Ito, Makoto; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2015-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.

  12. Na+/H+ exchanger 1 participates in tobacco disease defence against Phytophthora parasitica var. nicotianae by affecting vacuolar pH and priming the antioxidative system

    PubMed Central

    Chen, Xianyang; Bao, Hexigeduleng; Guo, Jie; Jia, Weitao; Tai, Fang; Nie, Lingling; Jiang, Ping; Feng, Juanjuan; Lv, Sulian; Li, Yinxin

    2014-01-01

    Despite the importance of NHX1 (Na+/H+ exchanger 1) in plant salt tolerance, little is known about its other functions. In this study, intriguingly, it was found that NHX1 participated in plant disease defence against Phytophthora parasitica var. nicotianae (Ppn) in Nicotiana benthamiana. NbNHX1 was originally isolated from N. benthamiana, and characterized. The subcellular localization of NbNHX1 with its C-terminus fused with green fluorescent protein indicated that NbNHX1 localized primarily to the tonoplast. Tobacco rattle virus-induced NbNHX1 silencing led to reduced H+ efflux from the vacuole to cytoplasts, and decreased Ppn resistance in N. benthamiana. After attack by Ppn, NbNHX1-silenced plants exhibited impaired ability to scavenge reactive oxidative species (ROS) induced by the pathogen. Pea early browning virus-mediated ectopic expression of SeNHX1 (from Salicornia europaea) or AtNHX1 (from Arabidopsis thaliana) both conferred enhanced Ppn resistance to N. benthamiana, with a lower H2O2 concentration after Ppn inoculation. Further investigation of the role of NHX1 demonstrated that transient overexpression of NbNHX1 improved the vacuolar pH and cellular ROS level in N. benthamiana, which was coupled with an enlarged NAD(P) (H) pool and higher expression of ROS-responsive genes. In contrast, NbNHX1 silencing led to a lower pH in the vacuole and a lower cellular ROS level in N. benthamiana, which was coupled with a decreased NAD(P) (H) pool and decreased expression of ROS-responsive genes. These results suggest that NHX1 is involved in plant disease defence; and regulation of vacuolar pH by NHX1, affecting the cellular oxidation state, primes the antioxidative system which is associated with Ppn resistance in tobacco. PMID:25170102

  13. MADS1, a novel MADS-box protein, is involved in the response of Nicotiana benthamiana to bacterial harpin(Xoo).

    PubMed

    Zhang, Huajian; Teng, Wenjun; Liang, Jingang; Liu, Xinyu; Zhang, Haifeng; Zhang, Zhengguang; Zheng, Xiaobo

    2016-01-01

    MADS-box transcription factor genes are well known for their role in floral organ and seed development. In this study, a novel MADS-box-containing gene, designated NbMADS1, was isolated from leaves of Nicotiana benthamiana. The full-length cDNA was 666 bp and encoded a putative polypeptide of 221 aa with a mass of 24.3 kDa. To assess the role of NbMADS1 in the defence response to bacterial harpin(Xoo), an elicitor of the hypersensitive response, a loss-of-function experiment was performed in N. benthamiana plants using virus-induced gene silencing. Analyses of electrolyte leakage revealed more extensive cell death in the control plants than in NbMADS1-silenced plants. The NbMADS1-silenced plants showed impaired harpin(Xoo)-induced stomatal closure, decreased harpin(Xoo)-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced harpin(Xoo)-induced resistance to Phytophthora nicotianae. The compromised stomatal closure observed in the NbMADS1-silenced plants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor). Taken together, these results demonstrate that the NbMADS1-H2O2-NO pathway mediates multiple harpin(Xoo)-triggered responses, including stomatal closure, hypersensitive cell death, and defence-related gene expression, suggesting that NbMADS1 plays an important role in regulating the response to harpin(Xoo) in N. benthamiana plants. PMID:26466663

  14. Expression of the cercosporin toxin resistance gene ( CRG1) as a dicistronic mRNA in the filamentous fungus Cercospora nicotianae.

    PubMed

    Chung, Kuang-Ren; Daub, Margaret E; Ehrenshaft, Marilyn

    2003-09-01

    The CRG1 gene in Cercospora nicotianae encodes a transcription factor and is required for cercosporin toxin resistance and production. Cloning and sequencing of the downstream region of the CRG1 gene led to the discovery of an adjacent gene ( PUT1) encoding a putative uracil transporter. Expression of CRG1 and PUT1 as assessed by Northern analysis indicated that, in addition to the expected monocistronic mRNAs (2.6 kb and 2.0 kb, respectively), a common 4.5-kb mRNA could be identified, using either a CRG1 or a PUT1 gene probe. The 2.6-kb transcript identified only by the CRG1 probe was expressed constitutively, whereas the 2.0-kb transcript identified only by the PUT1 probe was differentially expressed in various media. Four cDNA clones containing CRG1, PUT1, and the CRG1- PUT1 intergenic region were identified as part of the products from the 4.5-kb transcript. Both the 4.5-kb and 2.6-kb transcripts were not detectable in three crg1-disrupted mutants, using the CRG1 probe. The 2.0-kb transcript, but not the 4.5-kb one was detected using the PUT1 probe in the three crg1-disrupted mutants. Taken together, we conclude that the 4.5-kb transcript is a dicistronic mRNA of both CRG1 and PUT1 in the fungus C. nicotianae. This is the first example of a dicistronic mRNA identified in filamentous fungi.

  15. Cotton GhMKK5 affects disease resistance, induces HR-like cell death, and reduces the tolerance to salt and drought stress in transgenic Nicotiana benthamiana.

    PubMed

    Zhang, Liang; Li, Yuzhen; Lu, Wenjing; Meng, Fei; Wu, Chang-ai; Guo, Xingqi

    2012-06-01

    Mitogen-activated protein kinase (MAPK) cascades are involved in various processes from plant growth and development to biotic and abiotic stress responses. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), play crucial roles in MAPK cascades to mediate a variety of stress responses in plants. However, few MAPKKs have been functionally characterized in cotton (Gossypium hirsutum). In this study, a novel gene, GhMKK5, from cotton belonging to the group C MAPKKs was isolated and characterized. The expression of GhMKK5 can be induced by pathogen infection, abiotic stresses, and multiple defence-related signal molecules. The overexpression of GhMKK5 in Nicotiana benthamiana enhanced the plants' resistance to the bacterial pathogen Ralstonia solanacearum by elevating the expression of pathogen resistance (PR) genes, including PR1a, PR2, PR4, PR5, and NPR1, but increased the plants' sensitivity to the oomycete pathogen Phytophthora parasitica var. nicotianae Tucker. Importantly, GhMKK5-overexpressing plants displayed markedly elevated expression of reactive oxygen species-related and cell death marker genes, such as NtRbohA and NtCDM, and resulted in hypersensitive response (HR)-like cell death characterized by the accumulation of H(2)O(2). Furthermore, it was demonstrated that GhMKK5 overexpression in plants reduced their tolerance to salt and drought stresses, as determined by statistical analysis of seed germination, root length, leaf water loss, and survival rate. Drought obviously accelerated the cell death phenomenon in GhMKK5-overexpressing plants. These results suggest that GhMKK5 may play an important role in pathogen infection and the regulation of the salt and drought stress responses in plants.

  16. Transcriptomic and Reverse Genetic Analysesof Branched-Chain Fatty Acid and Acyl Sugar Production in Solanum pennellii and Nicotiana benthamiana1[W][OA

    PubMed Central

    Slocombe, Stephen P.; Schauvinhold, Ines; McQuinn, Ryan P.; Besser, Katrin; Welsby, Nicholas A.; Harper, Andrea; Aziz, Naveed; Li, Yi; Larson, Tony R.; Giovannoni, James; Dixon, Richard A.; Broun, Pierre

    2008-01-01

    Acyl sugars containing branched-chain fatty acids (BCFAs) are exuded by glandular trichomes of many species in Solanaceae, having an important defensive role against insects. From isotope-feeding studies, two modes of BCFA elongation have been proposed: (1) fatty acid synthase-mediated two-carbon elongation in the high acyl sugar-producing tomato species Solanum pennellii and Datura metel; and (2) α-keto acid elongation-mediated one-carbon increments in several tobacco (Nicotiana) species and a Petunia species. To investigate the molecular mechanisms underlying BCFAs and acyl sugar production in trichomes, we have taken a comparative genomic approach to identify critical enzymatic steps followed by gene silencing and metabolite analysis in S. pennellii and Nicotiana benthamiana. Our study verified the existence of distinct mechanisms of acyl sugar synthesis in Solanaceae. From microarray analyses, genes associated with α-keto acid elongation were found to be among the most strongly expressed in N. benthamiana trichomes only, supporting this model in tobacco species. Genes encoding components of the branched-chain keto-acid dehydrogenase complex were expressed at particularly high levels in trichomes of both species, and we show using virus-induced gene silencing that they are required for BCFA production in both cases and for acyl sugar synthesis in N. benthamiana. Functional analysis by down-regulation of specific KAS I genes and cerulenin inhibition indicated the involvement of the fatty acid synthase complex in BCFA production in S. pennellii. In summary, our study highlights both conserved and divergent mechanisms in the production of important defense compounds in Solanaceae and defines potential targets for engineering acyl sugar production in plants for improved pest tolerance. PMID:18931142

  17. Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum.

    PubMed

    Shitan, Nobukazu; Minami, Shota; Morita, Masahiko; Hayashida, Minaho; Ito, Shingo; Takanashi, Kojiro; Omote, Hiroshi; Moriyama, Yoshinori; Sugiyama, Akifumi; Goossens, Alain; Moriyasu, Masataka; Yazaki, Kazufumi

    2014-01-01

    Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues. PMID:25268729

  18. Involvement of the Leaf-Specific Multidrug and Toxic Compound Extrusion (MATE) Transporter Nt-JAT2 in Vacuolar Sequestration of Nicotine in Nicotiana tabacum

    PubMed Central

    Shitan, Nobukazu; Minami, Shota; Morita, Masahiko; Hayashida, Minaho; Ito, Shingo; Takanashi, Kojiro; Omote, Hiroshi; Moriyama, Yoshinori; Sugiyama, Akifumi; Goossens, Alain; Moriyasu, Masataka; Yazaki, Kazufumi

    2014-01-01

    Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues. PMID:25268729

  19. A Glycosyltransferase from Nicotiana alata Pollen Mediates Synthesis of a Linear (1,5)-α-L-Arabinan When Expressed in Arabidopsis1[OPEN

    PubMed Central

    Ho, Yin Ying; Moller, Isabel E.; Koh, Poh-Ling; Bacic, Antony

    2016-01-01

    The walls of Nicotiana alata pollen tubes contain a linear arabinan composed of (1,5)-α-linked arabinofuranose residues. Although generally found as a side chain on the backbone of the pectic polysaccharide rhamnogalacturonan I, the arabinan in N. alata pollen tubes is considered free, as there is no detectable rhamnogalacturonan I in these walls. Carbohydrate-specific antibodies detected arabinan epitopes at the tip and along the shank of N. alata pollen tubes that are predominantly part of the primary layer of the bilayered wall. A sequence related to ARABINAN DEFICIENT1 (AtARAD1), a presumed arabinan arabinosyltransferase from Arabidopsis (Arabidopsis thaliana), was identified by searching an N. alata pollen transcriptome. Transcripts for this ARAD1-like sequence, which we have named N. alata ARABINAN DEFICIENT-LIKE1 (NaARADL1), accumulate in various tissues, most abundantly in the pollen grain and tube, and encode a protein that is a type II membrane protein with its catalytic carboxyl terminus located in the Golgi lumen. The NaARADL1 protein can form homodimers when transiently expressed in Nicotiana benthamiana leaves and heterodimers when coexpressed with AtARAD1. The expression of NaARADL1 in Arabidopsis led to plants with more arabinan in their walls and that also exuded a guttation fluid rich in arabinan. Chemical and enzymatic characterization of the guttation fluid showed that a soluble, linear α-(1,5)-arabinan was the most abundant polymer present. These results are consistent with NaARADL1 having an arabinan (1,5)-α-arabinosyltransferase activity. PMID:26850276

  20. Differences in cell death induction by Phytophthora Elicitins are determined by signal components downstream of MAP kinase kinase in different species of Nicotiana and cultivars of Brassica rapa and Raphanus sativus.

    PubMed

    Takemoto, Daigo; Hardham, Adrienne R; Jones, David A

    2005-07-01

    Elicitins are small, secreted proteins produced by species of the plant-pathogenic oomycete Phytophthora. They induce hypersensitive cell death in most Nicotiana species and in some cultivars of Brassica rapa and Raphanus sativus. In this study, two true-breeding Fast Cycling B. rapa lines were established that showed severe necrosis (line 7-R) or no visible response (line 18-NR) after treatment with elicitin. Unexpectedly, microscopic examination revealed localized cell death in line 18-NR plants, and expression levels of various defense-marker genes were comparable in both lines. These results suggested that both "responsive" and "nonresponsive" plants responded to elicitin but differed in the extent of the cell death response. Expression of a constitutively active form of Arabidopsis (Arabidopsis thaliana) MAP kinase kinase 4 (AtMEK4(DD)) also induced rapid development of confluent cell death in line 7-R, whereas line 18-NR showed no visible cell death. Similarly, elicitin-responsive Nicotiana species and R. sativus cultivars showed significantly stronger cell death responses following expression of AtMEK4(DD) compared with nonresponsive species/cultivars. Line 7-R also showed higher sensitivity to toxin-containing culture filtrates produced by Alternaria brassicicola, and toxin sensitivity cosegregated with elicitin responsiveness, suggesting that the downstream responses induced by elicitin and Alternaria toxin share factors that control the extent of cell death. Interestingly, elicitin responsiveness was shown to correlate with greater susceptibility to A. brassicicola (a necrotroph) in B. rapa but less susceptibility to Phytophthora nicotianae (a hemibiotroph) in Nicotiana, suggesting a more extensive cell death response could cause opposite effects on the outcomes of biotrophic versus necrotrophic plant-pathogen interactions.

  1. Phenylpropanoid Defences in Nicotiana tabacum Cells: Overlapping Metabolomes Indicate Common Aspects to Priming Responses Induced by Lipopolysaccharides, Chitosan and Flagellin-22

    PubMed Central

    Mhlongo, Msizi I.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Steenkamp, Paul A.; Dubery, Ian A.

    2016-01-01

    Plants have evolved both constitutive and inducible defence strategies to cope with different biotic stimuli and stresses. Exposure of a plant to a challenging stress can lead to a primed state that allows it to launch a more rapid and stronger defence. Here we applied a metabolomic approach to study and compare the responses induced in Nicotiana tabacum cells by microbe-associated molecular pattern (MAMP) molecules, namely lipopolysaccharides (LPS), chitosan (CHT) and flagellin-22 (FLG22). Early response metabolites, extracted with methanol, were analysed by UHPLC-MS/MS. Using multivariate statistical tools the metabolic profiles induced by these elicitors were analysed. In the metabolic fingerprint of these agents a total of 19 cinnamic acid derivatives conjugated to quinic acids (chlorogenic acids), shikimic acid, tyramine, polyamines or glucose were found as discriminant biomarkers. In addition, treatment with the phytohormones salicylic acid (SA), methyljasmonic acid (MJ) and abscisic acid (ABA) resulted in differentially-induced phenylpropanoid pathway metabolites. The results indicate that the phenylpropanoid pathway is activated by these elicitors while hydroxycinnamic acid derivatives are commonly associated with the metabolic response to the MAMPs, and that the activated responses are modulated by both SA and MJ, with ABA not playing a role. PMID:26978774

  2. Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

    SciTech Connect

    Altabella, T.; Chrispeels, M.J. )

    1990-06-01

    Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{sub r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.

  3. Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies.

    PubMed

    Petre, Benjamin; Saunders, Diane G O; Sklenar, Jan; Lorrain, Cécile; Krasileva, Ksenia V; Win, Joe; Duplessis, Sébastien; Kamoun, Sophien

    2016-01-01

    Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors.

  4. Functional analyses of AGAMOUS family members in Nicotiana benthamiana clarify the evolution of early and late roles of C-function genes in eudicots.

    PubMed

    Fourquin, Chloé; Ferrándiz, Cristina

    2012-09-01

    The C-function, according to the ABC model of floral organ identity, is required for stamen and carpel development and to provide floral meristem determinacy. Members of the AG lineage of the large MADS box gene family specify the C-function in a broadly conserved manner in angiosperms. In core eudicots, two sub-lineages co-exist, euAG and PLE, which have been extensively characterized in Antirrhinum majus and Arabidopsis thaliana, where strong sub-functionalization has led to highly divergent contributions of the respective paralogs to the C-function. Various scenarios have been proposed to reconstruct the evolutionary history of the euAG and PLE lineages in eudicots, but detailed functional analyses of the roles of these genes in additional representative species to validate evolutionary hypotheses are scarce. Here, we report functional characterization of euAG- and PLE-like genes in Nicotiana benthamiana through expression analyses and phenotypic characterization of the defects caused by their specific down-regulation. We show that both paralogs redundantly contribute to the C-function in this species, providing insights on the likely evolution of these gene lineages following divergence of the major groups within the eudicots (rosids and asterids). Moreover, we have demonstrated a conserved role for the PLE-like genes in controlling fruit dehiscence, which strongly supports the ancestral role of PLE-like genes in late fruit development and suggests a common evolutionary origin of late developmental processes in dry (dehiscent) and fleshy (ripening) fruits.

  5. Metabolism of methoxychlor by the P450-monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after expression in cell cultures of Nicotiana tabacum.

    PubMed

    Joussen, Nicole; Schuphan, Ingolf; Schmidt, Burkhard

    2010-03-01

    Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

  6. Analysis of the enhancer-blocking function of the TBS element from Petunia hybrida in transgenic Arabidopsis thaliana and Nicotiana tabacum.

    PubMed

    Singer, Stacy D; Hily, Jean-Michel; Cox, Kerik D

    2011-11-01

    Transcriptional enhancers possess the ability to override the tissue-specificity and efficiency of nearby promoters, which is of concern when generating transgenic constructs bearing multiple cassettes. One means of preventing these inappropriate interactions is through the use of enhancer-blocking insulators. The 2-kb transformation booster sequence (TBS) from Petunia hybrida has been shown previously to exhibit this function when inserted between an enhancer and promoter in transgenic Arabidopsis thaliana. In this study, we attempted to further characterize the ability of this fragment to impede enhancer-promoter interference through an analysis of transgenic Arabidopsis and Nicotiana tabacum lines bearing various permutations of the TBS element between the cauliflower mosaic virus (CaMV) 35S enhancer and an assortment of tissue-specific promoters fused to the β-glucuronidase (GUS) reporter gene. The full-length TBS fragment was found to function in both orientations, although to a significantly lesser degree in the reverse orientation, and was operational in both plant species tested. While multiple deletion fragments were found to exhibit activity, it appeared that several regions of the TBS were required for maximal enhancer-blocking function. Furthermore, we found that this element exhibited promoter-like activity, which has implications in terms of possible mechanisms behind its ability to impede enhancer-promoter communication in plants.

  7. Nucleoporin 75 is involved in the ethylene-mediated production of phytoalexin for the resistance of Nicotiana benthamiana to Phytophthora infestans.

    PubMed

    Ohtsu, Mina; Shibata, Yusuke; Ojika, Makoto; Tamura, Kentaro; Hara-Nishimura, Ikuko; Mori, Hitoshi; Kawakita, Kazuhito; Takemoto, Daigo

    2014-12-01

    Mature Nicotiana benthamiana shows stable resistance to the oomycete pathogen Phytophthora infestans. Induction of phytoalexin (capsidiol) production is essential for the resistance, which is upregulated via a mitogen-activated protein kinase (MAPK) cascade (NbMEK2-WIPK/SIPK) followed by ethylene signaling. In this study, NbNup75 (encodes a nuclear pore protein Nucleoporin75) was identified as an essential gene for resistance of N. benthamiana to P. infestans. In NbNup75-silenced plants, initial events of elicitor-induced responses such as phosphorylation of MAPK and expression of defense-related genes were not affected, whereas induction of later defense responses such as capsidiol production and cell death induction was suppressed or delayed. Ethylene production induced by either INF1 or NbMEK2 was reduced in NbNup75-silenced plants, whereas the expression of NbEAS (a gene for capsidiol biosynthesis) induced by ethylene was not affected, indicating that Nup75 is required for the induction of ethylene production but not for ethylene signaling. Given that nuclear accumulation of polyA RNA was increased in NbNup75-silenced plants, efficient export of mRNA from nuclei via nuclear pores would be important for the timely upregulation of defense responses. Collectively, Nup75 is involved in the induction of a later stage of defense responses, including the ethylene-mediated production of phytoalexin for the resistance of N. benthamiana to P. infestans.

  8. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

    PubMed Central

    Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Yuan, Zheng; Zhang, Dabing

    2016-01-01

    Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS), and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise.

  9. N-Glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential

    PubMed Central

    Hamorsky, Krystal Teasley; Kouokam, J. Calvin; Jurkiewicz, Jessica M.; Nelson, Bailey; Moore, Lauren J.; Husk, Adam S.; Kajiura, Hiroyuki; Fujiyama, Kazuhito; Matoba, Nobuyuki

    2015-01-01

    Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants. PMID:25614217

  10. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway.

    PubMed

    Ritala, Anneli; Dong, Lemeng; Imseng, Nicole; Seppänen-Laakso, Tuulikki; Vasilev, Nikolay; van der Krol, Sander; Rischer, Heiko; Maaheimo, Hannu; Virkki, Arho; Brändli, Johanna; Schillberg, Stefan; Eibl, Regine; Bouwmeester, Harro; Oksman-Caldentey, Kirsi-Marja

    2014-04-20

    The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC-MS screening tool was developed for the rapid evaluation of production clones. The GC-MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 μg/g dry weight (DW) and a maximum of 31.3 μg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 μg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag. PMID:24530945

  11. The symbiosis between Nicotiana tabacum and the endomycorrhizal fungus Funneliformis mosseae increases the plant glutathione level and decreases leaf cadmium and root arsenic contents.

    PubMed

    Degola, Francesca; Fattorini, Laura; Bona, Elisa; Sprimuto, Christian Triscari; Argese, Emanuele; Berta, Graziella; Sanità di Toppi, Luigi

    2015-07-01

    Over time, anthropogenic activities have led to severe cadmium (Cd) and arsenic (As) pollution in several environments. Plants inhabiting metal(loid)-contaminated areas should be able to sequester and detoxify these toxic elements as soon as they enter roots and leaves. We postulated here that an important role in protecting plants from excessive metal(loid) accumulation and toxicity might be played by arbuscular mycorrhizal (AM) fungi. In fact, human exploitation of plant material derived from Cd- and As-polluted environments may lead to a noxious intake of these toxic elements; in particular, a possible source of Cd and As for humans is given by cigarette and cigar smoke. We investigated the role of AM fungus Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler in protecting Nicotiana tabacum L. (cv. Petit Havana) from the above-mentioned metal(loid) stress. Our findings proved that the AM symbiosis is effective in increasing the plant tissue content of the antioxidant glutathione (GSH), in influencing the amount of metal(loid)-induced chelators as phytochelatins, and in reducing the Cd and As content in leaves and roots of adult tobacco plants. These results might also prove useful in improving the quality of commercial tobacco, thus reducing the risks to human health due to inhalation of toxic elements contained in smoking products.

  12. Relationship between ozone, meteorological conditions, gas exchange and leaf injury in Nicotiana tabacum Bel-W3 in a sub-tropical region

    NASA Astrophysics Data System (ADS)

    Silva, Daiane T.; Meirelles, Sérgio T.; Moraes, Regina M.

    2012-12-01

    The city of São Paulo is located in a subtropical region whose climate exhibits few defined seasons as well as frequent oscillations in temperature and rainfall throughout the year. In addition to interfering with physiological processes, these peculiar climatic dynamics influence the formation of O3 and its influx into leaves, causing species used as bioindicators in temperate climates to be ineffective here. This study evaluated gas exchange variations in CO2 and H2O and leaf injuries induced by O3 in Nicotiana tabacum Bel-W3 in relation to oscillations in environmental conditions. Plants were exposed to an O3-polluted environment for fifteen periods of fourteen days each throughout 2008. Gas exchange and O3 were higher during the summer and winter but were highly variable in all seasons. Severe injuries occurred during the winter and spring, with significant variation in this parameter being observed throughout the year. An analysis of biotic and abiotic variables revealed complex relationships among them, with great importance of meteorological factors in plant responses. We conclude that under unstable climatic conditions, the relationship between O3 flux and injury is weak, and the qualitative character of biomonitoring is further confirmed.

  13. iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

    PubMed

    Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

    2016-01-15

    Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress. PMID:26692494

  14. Evidence for effects on the in vivo activity of ribulose-bisphosphate carboxylase/oxygenase during development of Mn toxicity in tobacco. [Nicotiana tabacum L. cv KY14

    SciTech Connect

    Houtz, R.L.; Nable, R.O.; Cheniae, G.M. )

    1988-04-01

    The progressive decrease in net photosynthesis accompanying development of Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) is a result of effects on in vivo activity of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (rubisco, EC 4.1.1.39). This conclusion is supported by: (a) decrease in rates of CO{sub 2} depletion during measurements of CO{sub 2} compensation, (b) increase in leaf RuBP concentrations, (c) progressive decreases in rate-constants of RuBP loss (light to dark transition analyses) with progressive increases of leaf Mn concentrations, and (d) restoration of diminished rates of net photosynthesis to control rates by elevated CO{sub 2} (5%). Moreover, elevated CO{sub 2} (1100 microliters per liter) during culture of Mn-treated plants decreased elevated RuBP concentrations to control levels and alleviated foliar symptoms of Mn toxicity. These effects of Mn toxicity on in vivo activity of rubisco were not expressed by in vitro kinetic analyses of rubisco prepared under conditions to sequester Mn or to adsorb polyphenols or their oxidation products. Similarly, the in vitro activity of fructose bisphosphatase (EC 3.1.3.11) was unaffected by Mn toxicity.

  15. Phenylpropanoid Defences in Nicotiana tabacum Cells: Overlapping Metabolomes Indicate Common Aspects to Priming Responses Induced by Lipopolysaccharides, Chitosan and Flagellin-22.

    PubMed

    Mhlongo, Msizi I; Piater, Lizelle A; Madala, Ntakadzeni E; Steenkamp, Paul A; Dubery, Ian A

    2016-01-01

    Plants have evolved both constitutive and inducible defence strategies to cope with different biotic stimuli and stresses. Exposure of a plant to a challenging stress can lead to a primed state that allows it to launch a more rapid and stronger defence. Here we applied a metabolomic approach to study and compare the responses induced in Nicotiana tabacum cells by microbe-associated molecular pattern (MAMP) molecules, namely lipopolysaccharides (LPS), chitosan (CHT) and flagellin-22 (FLG22). Early response metabolites, extracted with methanol, were analysed by UHPLC-MS/MS. Using multivariate statistical tools the metabolic profiles induced by these elicitors were analysed. In the metabolic fingerprint of these agents a total of 19 cinnamic acid derivatives conjugated to quinic acids (chlorogenic acids), shikimic acid, tyramine, polyamines or glucose were found as discriminant biomarkers. In addition, treatment with the phytohormones salicylic acid (SA), methyljasmonic acid (MJ) and abscisic acid (ABA) resulted in differentially-induced phenylpropanoid pathway metabolites. The results indicate that the phenylpropanoid pathway is activated by these elicitors while hydroxycinnamic acid derivatives are commonly associated with the metabolic response to the MAMPs, and that the activated responses are modulated by both SA and MJ, with ABA not playing a role. PMID:26978774

  16. Long-chain bases and their phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco (Nicotiana tabacum) BY-2 cells.

    PubMed

    Coursol, Sylvie; Fromentin, Jérôme; Noirot, Elodie; Brière, Christian; Robert, Franck; Morel, Johanne; Liang, Yun-Kuan; Lherminier, Jeannine; Simon-Plas, Françoise

    2015-02-01

    The proteinaceous elicitor cryptogein triggers defence reactions in Nicotiana tabacum (tobacco) through a signalling cascade, including the early production of reactive oxygen species (ROS) by the plasma membrane (PM)-located tobacco respiratory burst oxidase homologue D (NtRbohD). Sphingolipid long-chain bases (LCBs) are emerging as potent positive regulators of plant defence-related mechanisms. This led us to question whether both LCBs and their phosphorylated derivatives (LCB-Ps) are involved in the early signalling process triggered by cryptogein in tobacco BY-2 cells. Here, we showed that cryptogein-induced ROS production was inhibited by LCB kinase (LCBK) inhibitors. Additionally, Arabidopsis thaliana sphingosine kinase 1 and exogenously supplied LCB-Ps increased cryptogein-induced ROS production, whereas exogenously supplied LCBs had a strong opposite effect, which was not driven by a reduction in cellular viability. Immunogold-electron microscopy assay also revealed that LCB-Ps are present in the PM, which fits well with the presence of a high LCBK activity associated with this fraction. Our data demonstrate that LCBs and LCB-Ps differentially regulate cryptogein-induced ROS production in tobacco BY-2 cells, and support a model in which a cooperative synergism between LCBK/LCB-Ps and NtRbohD/ROS in the cryptogein signalling pathway is likely at the PM in tobacco BY-2 cells.

  17. The transmitting tissue of Nicotiana tabacum is not essential to pollen tube growth, and its ablation can reverse prezygotic interspecific barriers.

    PubMed

    Smith, Alan G; Eberle, Carrie A; Moss, Nicole G; Anderson, Neil O; Clasen, Benjamin M; Hegeman, Adrian D

    2013-12-01

    The Nicotiana tabacum transmitting tissue is a highly specialized file of metabolically active cells that is the pathway for pollen tubes from the stigma to the ovules where fertilization occurs. It is thought to be essential to pollen tube growth because of the nutrients and guidance it provides to the pollen tubes. It also regulates gametophytic self-incompatibility in the style. To test the function of the transmitting tissue in pollen tube growth and to determine its role in regulating prezygotic interspecific incompatibility, genetic ablation was used to eliminate the mature transmitting tissue, producing a hollow style. Despite the absence of the mature transmitting tissue and greatly reduced transmitting-tissue-specific gene expression, self-pollen tubes had growth to the end of the style. Pollen tubes grew at a slower rate in the transmitting-tissue-ablated line during the first 24 h post-pollination. However, pollen tubes grew to a similar length 40 h post-pollination with and without a transmitting tissue. Ablation of the N. tabacum transmitting tissue significantly altered interspecific pollen tube growth. These results implicate the N. tabacum transmitting tissue in facilitating or inhibiting interspecific pollen tube growth in a species-dependent manner and in controlling prezygotic reproductive barriers.

  18. Cloning and molecular characterisation of a Delta8-sphingolipid-desaturase from Nicotiana tabacum closely related to Delta6-acyl-desaturases.

    PubMed

    García-Maroto, Federico; Garrido-Cárdenas, José A; Michaelson, Louise V; Napier, Johnathan A; Alonso, Diego López

    2007-06-01

    Investigation on the absence of Delta(6)-desaturase activity in Nicotiana tabacum has led to the cloning of a new desaturase gene from this organism (NTDXDES) that exhibited unexpected biochemical activity. Cladistic analysis shows clustering of NTDXDES together with functional Delta(6)-acyl-desaturases of near Solanales plants, such as Borago and Echium. This group lies apart from that of previously characterised Delta(8)-sphingolipid-desaturases, which also includes two putative tobacco members identified in this study. Moreover, strong expression of NTDXDES is found in leaves, flowers, fruits and developing seeds of tobacco plants that is highly dependent on the development phase, with transcriptional activity being higher at stages of active tissue growth. This pattern is similar to that showed by Delta(6)-acyl-desaturases characterised in Boraginaceae species. However, functional assays using a yeast expression system revealed that the protein encoded by NTDXDES lacks Delta(6)-desaturase activity, but instead it is able to desaturate sphingolipid substrates by introducing a double bond on the Delta(8)-position. These data indicate that NTDXDES represent a novel desaturase gene placed in a different evolutionary lineage to that of previously characterised Delta(8)-desaturases. PMID:17325828

  19. Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity.

    PubMed

    Fernandez-del-Carmen, Asun; Juárez, Paloma; Presa, Silvia; Granell, Antonio; Orzáez, Diego

    2013-02-20

    The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins.

  20. Identification of amino acid residues of the coat protein of Sri Lankan cassava mosaic virus affecting symptom production and viral titer in Nicotiana benthamiana.

    PubMed

    Kelkar, Vaishali; Kushawaha, Akhilesh Kumar; Dasgupta, Indranil

    2016-06-01

    Sri Lankan cassava mosaic virus (SLCMV) is bipartite begomovirus infecting cassava in India and Sri Lanka. Interestingly, the DNA-A component of the SLCMV alone is able to infect Nicotiana benthamiana causing symptoms of upward leaf rolling and stunting. One of the differences between monopartite and bipartite begomoviruses is the requirement of Coat Protein (CP) for infectivity; CP being essential for the former, but dispensable in the latter. This investigation was aimed to determine the importance of CP in the infectivity of the bipartite SLCMV, behaving as a monopartite virus in N. benthamiana. We tested CP-null mutants, single amino acid replacement mutants and double, triple and quadruple combinations of the above in SLCMV DNA-A, for infectivity, symptom development and viral DNA accumulation in N. benthamiana. While CP-null mutants were non-infectious, a majority of the single amino acid replacement mutants and their combinations retained infectivity, some with attenuated symptoms and reduced viral titers. Some of the combined mutations restored the attenuated symptoms to wild type levels. Some of the mutations were predicted to cause changes in the secondary structure of the CP, which roughly correlated with the attenuation of symptoms and the reduction in viral titers. PMID:26948262

  1. Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases.

    PubMed

    Klinger, Johannes; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

  2. Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response.

    PubMed

    Van Cutsem, Emmanuel; Simonart, Géraldine; Degand, Hervé; Faber, Anne-Marie; Morsomme, Pierre; Boutry, Marc

    2011-02-01

    Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel-based and gel-free proteomic analyses were then performed. 2-DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI-MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2-DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense. PMID:21268273

  3. Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress.

    PubMed

    Schmidt, Gregor W; Delaney, Sven K

    2010-03-01

    Real-time RT-PCR is a powerful technique for the measurement of gene expression, but its accuracy depends on the stability of the internal reference gene(s) used for data normalization. Tobacco (Nicotiana tabacum) is an important model in studies of plant gene expression, but stable reference genes have not been well-studied in the tobacco system. We address this problem by analysing the expression stability of eight potential tobacco reference genes. Primers targeting each gene (18S rRNA, EF-1alpha, Ntubc2, alpha- and beta-tubulin, PP2A, L25 and actin) were developed and optimized. The expression of each gene was then measured by real-time PCR in a diverse set of 22 tobacco cDNA samples derived from developmentally distinct tissues and from plants exposed to several abiotic stresses. L25 and EF-1alpha demonstrated the highest expression stability, followed by Ntubc2. Measurement of L25 and EF-1alpha was sufficient for accurate normalization in either the developmental or stress-treated samples, but Ntubc2 was also required when considering the entire sample set. Analysis of a tobacco circadian gene (NTCP-23) verified these reference genes in an additional context, and all techniques were optimized to enable a high-throughput approach. These results provide a foundation for the more accurate and widespread use of real-time RT-PCR in tobacco. PMID:20098998

  4. Promotion of Bamboo Mosaic Virus Accumulation in Nicotiana benthamiana by 5′→3′ Exonuclease NbXRN4

    PubMed Central

    Lee, Cheng-Cheng; Lin, Tzu-Ling; Lin, Jhe-Wei; Han, Yu-Tsung; Huang, Yu-Ting; Hsu, Yau-Heiu; Meng, Menghsiao

    2016-01-01

    Bamboo mosaic virus (BaMV) has a 6.4-kb (+) sense RNA genome with a 5′ cap and a 3′ poly(A) tail. ORF1 of this potexvirus encodes a 155-kDa replication protein responsible for the viral RNA replication/transcription and 5′ cap formation. To learn more about the replication complex of BaMV, a protein preparation enriched in the 155-kDa replication protein was obtained from Nicotiana benthamiana by a protocol involving agroinfiltration and immunoprecipitation. Subsequent analysis by SDS-PAGE and mass spectrometry identified a handful of host proteins that may participate in the viral replication. Among them, the cytoplasmic exoribonuclease NbXRN4 particularly caught our attention. NbXRN4 has been shown to have an antiviral activity against Tomato bushy stunt virus and Tomato mosaic virus. In Arabidopsis, the enzyme could reduce RNAi- and miRNA-mediated RNA decay. This study found that downregulation of NbXRN4 greatly decreased BaMV accumulation, while overexpression of NbXRN4 resulted in an opposite effect. Mutations at the catalytically essential residues abolished the function of NbXRN4 in the increase of BaMV accumulation. Nonetheless, NbXRN4 was still able to promote BaMV accumulation in the presence of the RNA silencing suppressor P19. In summary, the replication efficiency of BaMV may be improved by the exoribonuclease activity of NbXRN4. PMID:26779163

  5. Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana.

    PubMed

    Kaido, Masanori; Inoue, Yosuke; Takeda, Yoshika; Sugiyama, Kazuhiko; Takeda, Atsushi; Mori, Masashi; Tamai, Atsushi; Meshi, Tetsuo; Okuno, Tetsuro; Mise, Kazuyuki

    2007-06-01

    The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.

  6. Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies.

    PubMed

    Petre, Benjamin; Saunders, Diane G O; Sklenar, Jan; Lorrain, Cécile; Krasileva, Ksenia V; Win, Joe; Duplessis, Sébastien; Kamoun, Sophien

    2016-01-01

    Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors. PMID:26863009

  7. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein

    PubMed Central

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  8. A three-dimensional RNA motif in Potato spindle tuber viroid mediates trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana.

    PubMed

    Takeda, Ryuta; Petrov, Anton I; Leontis, Neocles B; Ding, Biao

    2011-01-01

    Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, antiviral response, and cell-to-cell communication. A key mechanistic question is how an RNA is specifically selected for trafficking from one type of cell into another type. Here, we report the identification of an RNA motif in Potato spindle tuber viroid (PSTVd) required for trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana leaves. This motif, called loop 6, has the sequence 5'-CGA-3'...5'-GAC-3' flanked on both sides by cis Watson-Crick G/C and G/U wobble base pairs. We present a three-dimensional (3D) structural model of loop 6 that specifies all non-Watson-Crick base pair interactions, derived by isostericity-based sequence comparisons with 3D RNA motifs from the RNA x-ray crystal structure database. The model is supported by available chemical modification patterns, natural sequence conservation/variations in PSTVd isolates and related species, and functional characterization of all possible mutants for each of the loop 6 base pairs. Our findings and approaches have broad implications for studying the 3D RNA structural motifs mediating trafficking of diverse RNA species across specific cellular boundaries and for studying the structure-function relationships of RNA motifs in other biological processes.

  9. The symbiosis between Nicotiana tabacum and the endomycorrhizal fungus Funneliformis mosseae increases the plant glutathione level and decreases leaf cadmium and root arsenic contents.

    PubMed

    Degola, Francesca; Fattorini, Laura; Bona, Elisa; Sprimuto, Christian Triscari; Argese, Emanuele; Berta, Graziella; Sanità di Toppi, Luigi

    2015-07-01

    Over time, anthropogenic activities have led to severe cadmium (Cd) and arsenic (As) pollution in several environments. Plants inhabiting metal(loid)-contaminated areas should be able to sequester and detoxify these toxic elements as soon as they enter roots and leaves. We postulated here that an important role in protecting plants from excessive metal(loid) accumulation and toxicity might be played by arbuscular mycorrhizal (AM) fungi. In fact, human exploitation of plant material derived from Cd- and As-polluted environments may lead to a noxious intake of these toxic elements; in particular, a possible source of Cd and As for humans is given by cigarette and cigar smoke. We investigated the role of AM fungus Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler in protecting Nicotiana tabacum L. (cv. Petit Havana) from the above-mentioned metal(loid) stress. Our findings proved that the AM symbiosis is effective in increasing the plant tissue content of the antioxidant glutathione (GSH), in influencing the amount of metal(loid)-induced chelators as phytochelatins, and in reducing the Cd and As content in leaves and roots of adult tobacco plants. These results might also prove useful in improving the quality of commercial tobacco, thus reducing the risks to human health due to inhalation of toxic elements contained in smoking products. PMID:25900420

  10. RNA-sequencing Reveals Global Transcriptomic Changes in Nicotiana tabacum Responding to Topping and Treatment of Axillary-shoot Control Chemicals

    PubMed Central

    Singh, Sanjay K.; Wu, Yongmei; Ghosh, Jayadri S.; Pattanaik, Sitakanta; Fisher, Colin; Wang, Ying; Lawson, Darlene; Yuan, Ling

    2015-01-01

    Removal of terminal buds (topping) and control of the formation of axillary shoots (suckers) are common agronomic practices that significantly impact the yield and quality of various crop plants. Application of chemicals (suckercides) to plants following topping is an effective method for sucker control. However, our current knowledge of the influence of topping, and subsequent suckercide applications, to gene expression is limited. We analyzed the differential gene expression using RNA-sequencing in tobacco (Nicotiana tabacum) that are topped, or treated after topping by two different suckercides, the contact-localized-systemic, Flupro® (FP), and contact, Off-Shoot-T®. Among the differentially expressed genes (DEGs), 179 were identified as common to all three conditions. DEGs, largely related to wounding, phytohormone metabolism and secondary metabolite biosynthesis, exhibited significant upregulation following topping, and downregulation after suckercide treatments. DEGs related to photosynthetic processes were repressed following topping and suckercide treatments. Moreover, topping and FP-treatment affect the expression of auxin and cytokinin signaling pathway genes that are possibly involved in axillary shoot formation. Our results provide insights into the global change of plant gene expression in response to topping and suckercide treatments. The regulatory elements of topping-inducible genes are potentially useful for the development of a chemical-free sucker control system. PMID:26670135

  11. Virus-Induced Gene Silencing of Plastidial Soluble Inorganic Pyrophosphatase Impairs Essential Leaf Anabolic Pathways and Reduces Drought Stress Tolerance in Nicotiana benthamiana1[W][OA

    PubMed Central

    George, Gavin M.; van der Merwe, Margaretha J.; Nunes-Nesi, Adriano; Bauer, Rolene; Fernie, Alisdair R.; Kossmann, Jens; Lloyd, James R.

    2010-01-01

    The role of pyrophosphate in primary metabolism is poorly understood. Here, we report on the transient down-regulation of plastid-targeted soluble inorganic pyrophosphatase in Nicotiana benthamiana source leaves. Physiological and metabolic perturbations were particularly evident in chloroplastic central metabolism, which is reliant on fast and efficient pyrophosphate dissipation. Plants lacking plastidial soluble inorganic pyrophosphatase (psPPase) were characterized by increased pyrophosphate levels, decreased starch content, and alterations in chlorophyll and carotenoid biosynthesis, while constituents like amino acids (except for histidine, serine, and tryptophan) and soluble sugars and organic acids (except for malate and citrate) remained invariable from the control. Furthermore, translation of Rubisco was significantly affected, as observed for the amounts of the respective subunits as well as total soluble protein content. These changes were concurrent with the fact that plants with reduced psPPase were unable to assimilate carbon to the same extent as the controls. Furthermore, plants with lowered psPPase exposed to mild drought stress showed a moderate wilting phenotype and reduced vitality, which could be correlated to reduced abscisic acid levels limiting stomatal closure. Taken together, the results suggest that plastidial pyrophosphate dissipation through psPPase is indispensable for vital plant processes. PMID:20605913

  12. Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    PubMed Central

    Nemchinov, Lev G.; Boutanaev, Alexander M.; Postnikova, Olga A.

    2016-01-01

    In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development. PMID:27282827

  13. Virus-induced gene silencing of plastidial soluble inorganic pyrophosphatase impairs essential leaf anabolic pathways and reduces drought stress tolerance in Nicotiana benthamiana.

    PubMed

    George, Gavin M; van der Merwe, Margaretha J; Nunes-Nesi, Adriano; Bauer, Rolene; Fernie, Alisdair R; Kossmann, Jens; Lloyd, James R

    2010-09-01

    The role of pyrophosphate in primary metabolism is poorly understood. Here, we report on the transient down-regulation of plastid-targeted soluble inorganic pyrophosphatase in Nicotiana benthamiana source leaves. Physiological and metabolic perturbations were particularly evident in chloroplastic central metabolism, which is reliant on fast and efficient pyrophosphate dissipation. Plants lacking plastidial soluble inorganic pyrophosphatase (psPPase) were characterized by increased pyrophosphate levels, decreased starch content, and alterations in chlorophyll and carotenoid biosynthesis, while constituents like amino acids (except for histidine, serine, and tryptophan) and soluble sugars and organic acids (except for malate and citrate) remained invariable from the control. Furthermore, translation of Rubisco was significantly affected, as observed for the amounts of the respective subunits as well as total soluble protein content. These changes were concurrent with the fact that plants with reduced psPPase were unable to assimilate carbon to the same extent as the controls. Furthermore, plants with lowered psPPase exposed to mild drought stress showed a moderate wilting phenotype and reduced vitality, which could be correlated to reduced abscisic acid levels limiting stomatal closure. Taken together, the results suggest that plastidial pyrophosphate dissipation through psPPase is indispensable for vital plant processes.

  14. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

    PubMed

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  15. Exclusion of a Proton ATPase from the Apical Membrane Is Associated with Cell Polarity and Tip Growth in Nicotiana tabacum Pollen Tubes[W

    PubMed Central

    Certal, Ana C.; Almeida, Ricardo B.; Carvalho, Lara M.; Wong, Eric; Moreno, Nuno; Michard, Erwan; Carneiro, Jorge; Rodriguéz-Léon, Joaquín; Wu, Hen-Ming; Cheung, Alice Y.; Feijó, José A.

    2008-01-01

    Polarized growth in pollen tubes results from exocytosis at the tip and is associated with conspicuous polarization of Ca2+, H+, K+, and Cl− -fluxes. Here, we show that cell polarity in Nicotiana tabacum pollen is associated with the exclusion of a novel pollen-specific H+-ATPase, Nt AHA, from the growing apex. Nt AHA colocalizes with extracellular H+ effluxes, which revert to influxes where Nt AHA is absent. Fluorescence recovery after photobleaching analysis showed that Nt AHA moves toward the apex of growing pollen tubes, suggesting that the major mechanism of insertion is not through apical exocytosis. Nt AHA mRNA is also excluded from the tip, suggesting a mechanism of polarization acting at the level of translation. Localized applications of the cation ionophore gramicidin A had no effect where Nt AHA was present but acidified the cytosol and induced reorientation of the pollen tube where Nt AHA was absent. Transgenic pollen overexpressing Nt AHA-GFP developed abnormal callose plugs accompanied by abnormal H+ flux profiles. Furthermore, there is no net flux of H+ in defined patches of membrane where callose plugs are to be formed. Taken together, our results suggest that proton dynamics may underlie basic mechanisms of polarity and spatial regulation in growing pollen tubes. PMID:18364468

  16. Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies

    PubMed Central

    Petre, Benjamin; Saunders, Diane G. O.; Sklenar, Jan; Lorrain, Cécile; Krasileva, Ksenia V.; Win, Joe; Duplessis, Sébastien; Kamoun, Sophien

    2016-01-01

    Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors. PMID:26863009

  17. Cotton GhMKK1 Induces the Tolerance of Salt and Drought Stress, and Mediates Defence Responses to Pathogen Infection in Transgenic Nicotiana benthamiana

    PubMed Central

    Lu, Wenjing; Chu, Xiaoqian; Li, Yuzhen; Wang, Chen; Guo, Xingqi

    2013-01-01

    Mitogen-activated protein kinase kinases (MAPKK) mediate a variety of stress responses in plants. So far little is known on the functional role of MAPKKs in cotton. In the present study, Gossypium hirsutum MKK1 (GhMKK1) function was investigated. GhMKK1 protein may activate its specific targets in both the nucleus and cytoplasm. Treatments with salt, drought, and H2O2 induced the expression of GhMKK1 and increased the activity of GhMKK1, while overexpression of GhMKK1 in Nicotiana benthamiana enhanced its tolerance to salt and drought stresses as determined by many physiological data. Additionally, GhMKK1 activity was found to up-regulate pathogen-associated biotic stress, and overexpression of GhMKK1 increased the susceptibility of the transgenic plants to the pathogen Ralstonia solanacearum by reducing the expression of PR genes. Moreover, GhMKK1-overexpressing plants also exhibited an enhanced reactive oxygen species scavenging capability and markedly elevated activities of several antioxidant enzymes. These results indicate that GhMKK1 is involved in plants defence responses and provide new data to further analyze the function of plant MAPK pathways. PMID:23844212

  18. Rice salT promoter is activated in Papaver somniferum and Nicotiana tabacum transgenic cells in the absence of exogenous ABA.

    PubMed

    Elleuch; Belbahri; Boetti; David; Thomassetb; David

    2001-01-01

    With the aim of modifying secondary metabolism in Opium poppy (Papaver somniferum) and tobacco (Nicotiana tabacum) cells, gene transfer was performed using the sam1 gene from Arabidopsis thaliana under the control of the salT promoter. This promoter is induced by ABA in rice and in tobacco and we have shown that it is also induced in poppy cells (gus gene). Putatively transformed poppy and tobacco cell lines with the sam1 gene were obtained. In the absence of exogenous inducer we noticed the expression of the transgene resulting in a significant increase of SAM-S activity in all tested transformants of poppy and in half the transgenic tobacco cell lines tested. Addition of ABA to the culture medium failed to enhance the expression of the transgene in both species and resulted in a decrease of the sam1 gene expression in some cell lines. Since the salT promoter is induced by exogenous ABA in both species (gus reporter gene), we suggest a partial sam1 transgene inactivation in certain cell lines. These results show that the efficiency of a regulatory sequence may be different when fused with a reporter gene (gus) compared to fusion with a gene belonging to the housekeeping family (sam1).

  19. The 1.55 A resolution structure of Nicotiana alata S(F11)-RNase associated with gametophytic self-incompatibility.

    PubMed

    Ida, K; Norioka, S; Yamamoto, M; Kumasaka, T; Yamashita, E; Newbigin, E; Clarke, A E; Sakiyama, F; Sato, M

    2001-11-16

    The crystal structure of Nicotiana alata (ornamental tobacco) S(F11)-RNase, an S-allelic glycoprotein associated with gametophytic self-incompatibility, was determined by X-ray diffraction at 1.55 A resolution. The protein has a tertiary structure typical of members of the RNase T(2) family as it consists of a variant of the (alpha+beta) fold and has eight helices and seven strands. A heptasaccharide moiety is also present, and amino acid residues that serve as the catalytic acid and base can be assigned to His32 and His91, respectively. Two "hypervariable" regions, known as HVa and HVb, are the proposed sites of S-allele discrimination during the self-incompatibility reaction, and in the S(F11)-RNase these are well separated from the active site. HVa and HVb are composed of a long, positively charged loop followed by a part of an alpha-helix and short, negatively charged alpha-helix, respectively. The S(F11)-RNase structure shows both regions are readily accessible to the solvent and hence could participate in the process of self/non-self discrimination between the S-RNase and an unknown pollen S-gene product(s) upon pollination. PMID:11724536

  20. RNA-sequencing Reveals Global Transcriptomic Changes in Nicotiana tabacum Responding to Topping and Treatment of Axillary-shoot Control Chemicals.

    PubMed

    Singh, Sanjay K; Wu, Yongmei; Ghosh, Jayadri S; Pattanaik, Sitakanta; Fisher, Colin; Wang, Ying; Lawson, Darlene; Yuan, Ling

    2015-01-01

    Removal of terminal buds (topping) and control of the formation of axillary shoots (suckers) are common agronomic practices that significantly impact the yield and quality of various crop plants. Application of chemicals (suckercides) to plants following topping is an effective method for sucker control. However, our current knowledge of the influence of topping, and subsequent suckercide applications, to gene expression is limited. We analyzed the differential gene expression using RNA-sequencing in tobacco (Nicotiana tabacum) that are topped, or treated after topping by two different suckercides, the contact-localized-systemic, Flupro(®) (FP), and contact, Off-Shoot-T(®). Among the differentially expressed genes (DEGs), 179 were identified as common to all three conditions. DEGs, largely related to wounding, phytohormone metabolism and secondary metabolite biosynthesis, exhibited significant upregulation following topping, and downregulation after suckercide treatments. DEGs related to photosynthetic processes were repressed following topping and suckercide treatments. Moreover, topping and FP-treatment affect the expression of auxin and cytokinin signaling pathway genes that are possibly involved in axillary shoot formation. Our results provide insights into the global change of plant gene expression in response to topping and suckercide treatments. The regulatory elements of topping-inducible genes are potentially useful for the development of a chemical-free sucker control system. PMID:26670135

  1. Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses.

    PubMed

    Scalabrin, Elisa; Radaelli, Marta; Capodaglio, Gabriele

    2016-06-01

    The presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgenes.

  2. Ectopic expression of Capsicum-specific cell wall protein Capsicum annuum senescence-delaying 1 (CaSD1) delays senescence and induces trichome formation in Nicotiana benthamiana.

    PubMed

    Seo, Eunyoung; Yeom, Seon-In; Jo, Sunghwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

    2012-04-01

    Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.

  3. N-glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential.

    PubMed

    Hamorsky, Krystal Teasley; Kouokam, J Calvin; Jurkiewicz, Jessica M; Nelson, Bailey; Moore, Lauren J; Husk, Adam S; Kajiura, Hiroyuki; Fujiyama, Kazuhito; Matoba, Nobuyuki

    2015-01-23

    Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

  4. Comparison of small RNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot tospovirus reveals host-specific responses to viral infection.

    PubMed

    Margaria, Paolo; Miozzi, Laura; Ciuffo, Marina; Rosa, Cristina; Axtell, Michael J; Pappu, Hanu R; Turina, Massimo

    2016-01-01

    Viral small RNAs (vsRNAs) are one of the key elements involved in RNA silencing-based defense against viruses in plants. We analyzed the vsRNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot virus (PolRSV) (Tospovirus, Bunyaviridae). VsRNAs were abundant in both hosts, but a different size profile was observed, with an abundance peak at 21 in N. benthamiana and at 22 nt in tomato. VsRNAs mapping to the PolRSV L genomic segment were under-represented in both hosts, while S and M segments were differentially and highly targeted in N. benthamiana and tomato, respectively. Differences in preferential targeting of single ORFs were observed, with over-representation of NSs ORF-derived reads in N. benthamiana. Intergenic regions (IGRs)-mapping vsRNAs were under-represented, while enrichment of vsRNAs reads mapping to the NSs positive sense strand was observed in both hosts. Comparison with a previous study on tomato spotted wilt virus (TSWV) under the same experimental conditions, showed that the relative accumulation of PolRSV-specific and endogenous sRNAs was similar to the one observed for silencing suppressor-deficient TSWV strains, suggesting possible different properties of PolRSV NSs silencing suppressor compared to that of TSWV. PMID:26432447

  5. Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases

    PubMed Central

    Klinger, Johannes; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host. PMID:26648951

  6. The Arabidopsis LecRK-VI.2 associates with the pattern-recognition receptor FLS2 and primes Nicotiana benthamiana pattern-triggered immunity.

    PubMed

    Huang, Pin-Yao; Yeh, Yu-Hung; Liu, An-Chi; Cheng, Chiu-Ping; Zimmerli, Laurent

    2014-07-01

    Pattern-triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad-spectrum disease resistance. PTI is activated upon perception of microbe-associated molecular patterns (MAMPs) by pattern-recognition receptors (PRRs). We have recently demonstrated that the L-type lectin receptor kinase-VI.2 (LecRK-VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull-down, bimolecular fluorescence complementation and co-immunoprecipitation analyses that LecRK-VI.2 associates with the PRR FLS2. We also demonstrated that LecRK-VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK-VI.2 were indeed more resistant to virulent hemi-biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK-VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK-VI.2 in N. benthamiana primed PTI-mediated reactive oxygen species production, mitogen-activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK-VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.

  7. Identification of amino acid residues of the coat protein of Sri Lankan cassava mosaic virus affecting symptom production and viral titer in Nicotiana benthamiana.

    PubMed

    Kelkar, Vaishali; Kushawaha, Akhilesh Kumar; Dasgupta, Indranil

    2016-06-01

    Sri Lankan cassava mosaic virus (SLCMV) is bipartite begomovirus infecting cassava in India and Sri Lanka. Interestingly, the DNA-A component of the SLCMV alone is able to infect Nicotiana benthamiana causing symptoms of upward leaf rolling and stunting. One of the differences between monopartite and bipartite begomoviruses is the requirement of Coat Protein (CP) for infectivity; CP being essential for the former, but dispensable in the latter. This investigation was aimed to determine the importance of CP in the infectivity of the bipartite SLCMV, behaving as a monopartite virus in N. benthamiana. We tested CP-null mutants, single amino acid replacement mutants and double, triple and quadruple combinations of the above in SLCMV DNA-A, for infectivity, symptom development and viral DNA accumulation in N. benthamiana. While CP-null mutants were non-infectious, a majority of the single amino acid replacement mutants and their combinations retained infectivity, some with attenuated symptoms and reduced viral titers. Some of the combined mutations restored the attenuated symptoms to wild type levels. Some of the mutations were predicted to cause changes in the secondary structure of the CP, which roughly correlated with the attenuation of symptoms and the reduction in viral titers.

  8. Changes in polyphenol and sugar concentrations in wild type and genetically modified Nicotiana langsdorffii Weinmann in response to water and heat stress.

    PubMed

    Ancillotti, Claudia; Bogani, Patrizia; Biricolti, Stefano; Calistri, Elisa; Checchini, Leonardo; Ciofi, Lorenzo; Gonnelli, Cristina; Del Bubba, Massimo

    2015-12-01

    In this study wild type Nicotiana langsdorffii plants were genetically transformed by the insertion of the rat gene (gr) encoding the glucocorticoid receptor or the rolC gene and exposed to water and heat stress. Water stress was induced for 15 days by adding 20% PEG 6000 in the growth medium, whereas the heat treatment was performed at 50 °C for 2 h, after that a re-growing capability study was carried out. The plant response to stress was investigated by determining electrolyte leakage, dry weight biomass production and water content. These data were evaluated in relation to antiradical activity and concentrations of total polyphenols, selected phenolic compounds and some soluble sugars, as biochemical indicators of metabolic changes due to gene insertion and/or stress treatments. As regards the water stress, the measured physiological parameters evidenced an increasing stress level in the order rolC < gr < WT plants (e.g. about 100% and 50% electrolyte leakage increase in WT and gr samples, respectively) and complied with the biochemical pattern, which consisted in a general decrease of antiradical activity and phenolics, together with an increase in sugars. As regard heat stress, electrolyte leakage data were only in partial agreement with the re-growing capability study. In fact, according to this latter evaluation, gr was the genotype less affected by the heat shock. In this regard, sugars and especially phenolic compounds are informative of the long-term effects due to heat shock treatment.

  9. Investigation of the mechanism of nicotine demethylation in Nicotiana through 2H and 15N heavy isotope effects: implication of cytochrome P450 oxidase and hydroxyl ion transfer.

    PubMed

    Molinié, Roland; Kwiecień, Renata A; Paneth, Piotr; Hatton, Wilfried; Lebreton, Jacques; Robins, Richard J

    2007-02-15

    Heavy-atom isotope effects for the N-demethylation of nicotine have been determined in vivo in static-phase biosynthetically incompetent plant cell cultures of Nicotiana species. A (2)H kinetic isotope effect of 0.587 and a (15)N kinetic isotope effect of 1.0028 were obtained. An identical (15)N kinetic isotope effect of 1.0032 was obtained for the nicotine analogue, N-methyl-2-phenylpyrrolidine. The magnitude of the (15)N heavy-atom isotope effect indicates that the fission of the CN bond is not rate limiting for demethylation. The theoretical calculation of heavy-atom isotope effects for a model of the reaction pathway based on cytochrome P450 best fits the measured kinetic isotope effect to the addition of hydroxyl ion to iminium to form N-hydroxymethyl, for which the computed (2)H- and (15)N kinetic isotope effects are 0.689 and 1.0081, respectively. This large inverse (2)H kinetic isotope effect is not compatible with the initial abstraction of the H from the methyl group playing a significant kinetic role in the overall kinetic limitation of the reaction pathway, since computed values for this step (4.54 and 0.9995, respectively) are inconsistent with the experimental data.

  10. The unfolded protein response and programmed cell death are induced by expression of Garlic virus X p11 in Nicotiana benthamiana.

    PubMed

    Lu, Yuwen; Yin, Mingyuan; Wang, Xiaodan; Chen, Binghua; Yang, Xue; Peng, Jiejun; Zheng, Hongying; Zhao, Jinping; Lin, Lin; Yu, Chulang; MacFarlane, Stuart; He, Jianqing; Liu, Yong; Chen, Jianping; Dai, Liangying; Yan, Fei

    2016-06-01

    Garlic virus X (GarVX) ORF3 encodes a p11 protein, which contributes to virus cell-to-cell movement and forms granules on the endoplasmic reticulum (ER) in Nicotiana benthamiana. Expression of p11 either from a binary vector, PVX or TMV induced ER stress and the unfolded protein response (UPR), as demonstrated by an increase in transcription of the ER luminal binding protein (BiP) and bZIP60 genes. UPR-related programmed cell death (PCD) was elicited by PVX : p11 or TMV : p11 in systemic infected leaves. Examination of p11 mutants with deletions of two transmembrane domains (TM) revealed that both were required for generating granules and for inducing necrosis. TRV-based VIGS was used to investigate the correlation between bZIP60 expression and p11-induced UPR-related PCD. Less necrosis was observed on local and systemic leaves of bZIP60 knockdown plants when infected with PVXp11, suggesting that bZIP60 plays an important role in the UPR-related PCD response to p11 in N. benthamiana. PMID:27011387

  11. iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

    PubMed

    Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

    2016-01-15

    Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress.

  12. Feruloylputrescine and Caffeoylputrescine Are Not Involved in Growth and Floral Bud Formation of Stem Explants from Nicotiana tabacum L. var Xanthi nc

    PubMed Central

    Wyss-Benz, Markus; Streit, Luc; Ebert, Edith

    1990-01-01

    The role of feruloylputrescine (FP) and of caffeoylputrescine (CP) was investigated in an explant system of stem explants from day-neutral Nicotiana tabacum L. var Xanthi nc. Previously, a correlation between cortical callus formation and increase in FP content, as well as between in vitro flower formation and increase in CP content had been shown. During the explant growth in vitro, the increase of both FP and CP was inhibited by 4-fluor-(1-amino-2-phenylethyl)phosphonic acid and 2-amino-indene-2-phosphonic acid, both inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5). dl-α-difluoromethylarginine, an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), prevented only the increase in FP, while dl-α-difluoromethylornithine, an inhibitor of ornithine decarboxylase (EC 4.1.1.17), reduced only that of CP. Increase in dry weight and the formation of cortical callus and of floral buds of explants were not affected by any of the inhibitors. We conclude, in contrast to earlier hypotheses, that FP and CP do not trigger growth and differentiation in the explants. It seems more likely that FP and CP increase in response to auxin and cytokinin in the media. Images Figure 1 PMID:16667407

  13. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

    PubMed Central

    Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Yuan, Zheng; Zhang, Dabing

    2016-01-01

    Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS), and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise. PMID:27635142

  14. A Three-Dimensional RNA Motif in Potato spindle tuber viroid Mediates Trafficking from Palisade Mesophyll to Spongy Mesophyll in Nicotiana benthamiana[W

    PubMed Central

    Takeda, Ryuta; Petrov, Anton I.; Leontis, Neocles B.; Ding, Biao

    2011-01-01

    Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, antiviral response, and cell-to-cell communication. A key mechanistic question is how an RNA is specifically selected for trafficking from one type of cell into another type. Here, we report the identification of an RNA motif in Potato spindle tuber viroid (PSTVd) required for trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana leaves. This motif, called loop 6, has the sequence 5′-CGA-3′...5′-GAC-3′ flanked on both sides by cis Watson-Crick G/C and G/U wobble base pairs. We present a three-dimensional (3D) structural model of loop 6 that specifies all non-Watson-Crick base pair interactions, derived by isostericity-based sequence comparisons with 3D RNA motifs from the RNA x-ray crystal structure database. The model is supported by available chemical modification patterns, natural sequence conservation/variations in PSTVd isolates and related species, and functional characterization of all possible mutants for each of the loop 6 base pairs. Our findings and approaches have broad implications for studying the 3D RNA structural motifs mediating trafficking of diverse RNA species across specific cellular boundaries and for studying the structure-function relationships of RNA motifs in other biological processes. PMID:21258006

  15. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products.

    PubMed

    Biswas, Sukumar; Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Kok, Esther; Yuan, Zheng; Zhang, Dabing; Shi, Jianxin

    2016-01-01

    Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5'-monophosphate synthase (UMPS), and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise. PMID:27635142

  16. Overexpression of GhWRKY27a reduces tolerance to drought stress and resistance to Rhizoctonia solani infection in transgenic Nicotiana benthamiana

    PubMed Central

    Yan, Yan; Jia, Haihong; Wang, Fang; Wang, Chen; Liu, Shuchang; Guo, Xingqi

    2015-01-01

    WRKY proteins constitute transcriptional regulators involved in various biological processes, especially in coping with diverse biotic and abiotic stresses. However, in contrast to other well-characterized WRKY groups, the functions of group III WRKY transcription factors are poorly understood in the economically important crop cotton (Gossypium hirsutum). In this study, a group III WRKY gene from cotton, GhWRKY27a, was isolated and characterized. Our data indicated that GhWRKY27a localized to the nucleus and that GhWRKY27a expression could be strongly induced by abiotic stresses, pathogen infection, and multiple defense-related signaling molecules. Virus-induced gene silencing (VIGS) of GhWRKY27a enhanced tolerance to drought stress in cotton. In contrast, GhWRKY27a overexpression in Nicotiana benthamiana markedly reduced plant tolerance to drought stress, as determined through physiological analyses of leaf water loss, survival rates, and the stomatal aperture. This susceptibility was coupled with reduced stomatal closure in response to abscisic acid and decreased expression of stress-related genes. In addition, GhWRKY27a-overexpressing plants exhibited reduced resistance to Rhizoctonia solani infection, mainly demonstrated by the transgenic lines exhibiting more severe disease symptoms, accompanied by attenuated expression of defense-related genes in N. benthamiana. Taken together, these findings indicated that GhWRKY27a functions in negative responses to drought tolerance and in resistance to R. solani infection. PMID:26483697

  17. The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90)

    PubMed Central

    Zhang, Hongbo

    2014-01-01

    Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes. PMID:24604735

  18. Functional validation of Capsicum frutescens aminotransferase gene involved in vanillylamine biosynthesis using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures.

    PubMed

    Gururaj, Harishchandra B; Padma, Mallaya N; Giridhar, Parvatam; Ravishankar, Gokare A

    2012-10-01

    Capsaicinoid biosynthesis involves the participation of two substrates viz. vanillylamine and C(9)-C(11) fatty acid moieties. Vanillylamine which is a derivative of vanillin is synthesized through a transaminase reaction in the phenylpropanoid pathway of capsaicinoid synthesis. Here we report the functional validation of earlier reported putative aminotransferase gene for vanillylamine biosynthesis in heterologous system using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures. Molecular analysis tools comprising PCR and Southern blot analysis have shown the integration of the foreign gene in N. tabacum and C. frutescens calli cultures. The study shows the production of vanillylamine in transformed N. tabacum callus cultures and also the reduction of vanillylamine production when whole gene based antisense binary vector construct was used in transformation of C. frutescens callus cultures. Vanillylamine production, aminotransferase assay with Western blot analysis for crude proteins of transformants established the production of putative aminotransferase (pAMT) protein in alternate plant. The result is a clear evidence of involvement of the reported putative aminotransferase responsible for vanillylamine biosynthesis in capsaicinoid biosynthesis pathway, confirming the gene function through functional validation.

  19. Expression of ipt gene controlled by an ethylene and auxin responsive fragment of the LEACO1 promoter increases flower number in transgenic Nicotiana tabacum.

    PubMed

    Khodakovskaya, Mariya; Zhao, Degang; Smith, William; Li, Yi; McAvoy, Richard

    2006-11-01

    Cytokinins play important roles in regulating plant growth and development. A new genetic construct for regulating cytokinin content in plant cells was cloned and tested. The gene coding for isopentenyl transferase (ipt) was placed under the control of a 0.821 kb fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene promoter from Lycopersicon esculentum (LEACO1) and introduced into Nicotiana tabacum (cv. Havana). Some LEACO1(0.821) (kb)-ipt transgenic plant lines displayed normal shoot morphology but with a dramatic increase in the number of flower buds compared to nontransgenic plants. Other transgenic lines produced excessive lateral branch development but no change in flower bud number. Isolated leaves of transgenic tobacco plants showed a significantly prolonged retention of chlorophyll under dark incubation (25 degrees C for 20 days). Leaves of nontransformed plants senesced gradually under the same conditions. Experiments with LEACO1(0.821) (kb)-gus transgenic tobacco plants suggested auxin and ethylene involvement in induction of LEACO1(0.821) (kb) promoter activity. Multiple copies of nucleotide base sequences associated with either ethylene or auxin response elements were identified in the LEACO1(0.821) (kb) promoter fragment. The LEACO1(0.821) (kb)-ipt fusion gene appears to have potential utility for improving certain ornamental and agricultural crop species by increasing flower bud initiation and altering branching habit. PMID:16786314

  20. Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

    PubMed Central

    Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

    2016-01-01

    Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1–0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. PMID:26969746

  1. Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion[W

    PubMed Central

    Ischebeck, Till; Stenzel, Irene; Heilmann, Ingo

    2008-01-01

    Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P2 production at that location are uncharacterized, functions of PtdIns(4,5)P2 in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P2 production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P2 production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P2 via regulatory effects on membrane trafficking and/or apical pectin deposition. PMID:19060112

  2. Changes in polyphenol and sugar concentrations in wild type and genetically modified Nicotiana langsdorffii Weinmann in response to water and heat stress.

    PubMed

    Ancillotti, Claudia; Bogani, Patrizia; Biricolti, Stefano; Calistri, Elisa; Checchini, Leonardo; Ciofi, Lorenzo; Gonnelli, Cristina; Del Bubba, Massimo

    2015-12-01

    In this study wild type Nicotiana langsdorffii plants were genetically transformed by the insertion of the rat gene (gr) encoding the glucocorticoid receptor or the rolC gene and exposed to water and heat stress. Water stress was induced for 15 days by adding 20% PEG 6000 in the growth medium, whereas the heat treatment was performed at 50 °C for 2 h, after that a re-growing capability study was carried out. The plant response to stress was investigated by determining electrolyte leakage, dry weight biomass production and water content. These data were evaluated in relation to antiradical activity and concentrations of total polyphenols, selected phenolic compounds and some soluble sugars, as biochemical indicators of metabolic changes due to gene insertion and/or stress treatments. As regards the water stress, the measured physiological parameters evidenced an increasing stress level in the order rolC < gr < WT plants (e.g. about 100% and 50% electrolyte leakage increase in WT and gr samples, respectively) and complied with the biochemical pattern, which consisted in a general decrease of antiradical activity and phenolics, together with an increase in sugars. As regard heat stress, electrolyte leakage data were only in partial agreement with the re-growing capability study. In fact, according to this latter evaluation, gr was the genotype less affected by the heat shock. In this regard, sugars and especially phenolic compounds are informative of the long-term effects due to heat shock treatment. PMID:26410575

  3. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    PubMed

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. PMID

  4. Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L).

    PubMed

    Srinivasan, C; Liu, Zongrang; Scorza, Ralph

    2011-04-01

    Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.

  5. Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines.

    PubMed

    Nelson, Guillermo; Marconi, Patricia; Periolo, Osvaldo; La Torre, José; Alvarez, María Alejandra

    2012-06-22

    The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest inductor of the immune response. There are several available commercial vaccines against bovine viral diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts. An estimated amount of 20 μg of tE2 per gram of fresh leaves was regularly obtained with this plant system. Injection of guinea pigs with plant extracts containing 20 μg of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines. This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the advantage to be free of any eventual animal contaminant. PMID:22554468

  6. Nicotiana plumbaginifolia hlg mutants have a mutation in a PHYB-type phytochrome gene: they have elongated hypocotyls in red light, but are not elongated as adult plants.

    PubMed

    Hudson, M; Robson, P R; Kraepiel, Y; Caboche, M; Smith, H

    1997-11-01

    Two new allelic mutants of Nicotiana plumbaginifolia have been isolated which display a hypocotyl which is long (hlg) when seedlings are grown in continuous white light (W). This can be accounted for by the decreased response to red light (R) of the hypocotyl elongation rate in these mutants. Responses to other wavelengths are unaffected in the mutants. When grown in white light, mature hlg mutants are not elongated with respect to the wild-type; they also bolt and flower later. The shade-avoidance responses to red/far red ratio (R:FR) are intact in these mutants. Both mutants are deficient in phyB-like polypeptide that is immunodetectable in the wild-type; both have wild-type levels of a phyA-like polypeptide. These alleles are inherited in a partially dominant manner, and correspond to single-base missense mutations in a gene highly homologous to N. tabacum PHYB, which codes for a phytochrome B-type photoreceptor. One allele, hlg-1, has an introduced amino acid substitution; this may define a residue essential for phytochrome protein stability. The other allele, hlg-2, has a stop codon introduced C-terminal to the chromophore binding domain. As these phyB mutants are unaffected in shade-avoidance responses, but deficient in perception of R, it is concluded that the phyB absent in these mutants is responsible for R perception in the N. plumbaginifolia seedling, but is not a R:FR sensor in light-grown plants.

  7. Overexpression of SpWRKY1 promotes resistance to Phytophthora nicotianae and tolerance to salt and drought stress in transgenic tobacco.

    PubMed

    Li, Jing-bin; Luan, Yu-shi; Liu, Zhen

    2015-11-01

    WRKY transcription factors are key regulatory components of plant responses to biotic and abiotic stresses. SpWRKY1, a pathogen-induced WRKY gene, was isolated from tomato (Solanum pimpinellifolium L3708) using in silico cloning and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. SpWRKY1 expression was significantly induced following oomycete pathogen infection and treatment with salt, drought, salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA). Overexpression of SpWRKY1 in tobacco conferred greater resistance to Phytophthora nicotianae infection, as evidenced by lower malondialdehyde (MDA) content; relative electrolyte leakage (REL); higher chlorophyll content; and higher peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activities. This resistance was also coupled with enhanced expression of SA- and JA-associated genes (NtPR1, NtPR2, NtPR4, NtPR5 and NtPDF1.2), as well as of various defense-related genes (NtPOD, NtSOD and NtPAL). In addition, transgenic tobacco plants also displayed an enhanced tolerance to salt and drought stresses, mainly demonstrated by the transgenic lines exhibiting lower accumulation of MDA content and higher POD (EC 1.11.1.7), SOD (EC 1.15.1.1) activities, chlorophyll content, photosynthetic rate and stomatal conductance, accompanied by enhanced expression of defense-related genes (NtPOD, NtSOD, NtLEA5, NtP5CS and NtNCED1) under salt and drought stresses. Overall, these findings suggest that SpWRKY1 acts as a positive regulator involved in tobacco defense responses to biotic and abiotic stresses. PMID:25496091

  8. Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.

    PubMed

    Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

    2016-04-01

    Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. PMID:26969746

  9. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    PubMed

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

  10. Overexpression of SpWRKY1 promotes resistance to Phytophthora nicotianae and tolerance to salt and drought stress in transgenic tobacco.

    PubMed

    Li, Jing-bin; Luan, Yu-shi; Liu, Zhen

    2015-11-01

    WRKY transcription factors are key regulatory components of plant responses to biotic and abiotic stresses. SpWRKY1, a pathogen-induced WRKY gene, was isolated from tomato (Solanum pimpinellifolium L3708) using in silico cloning and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. SpWRKY1 expression was significantly induced following oomycete pathogen infection and treatment with salt, drought, salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA). Overexpression of SpWRKY1 in tobacco conferred greater resistance to Phytophthora nicotianae infection, as evidenced by lower malondialdehyde (MDA) content; relative electrolyte leakage (REL); higher chlorophyll content; and higher peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activities. This resistance was also coupled with enhanced expression of SA- and JA-associated genes (NtPR1, NtPR2, NtPR4, NtPR5 and NtPDF1.2), as well as of various defense-related genes (NtPOD, NtSOD and NtPAL). In addition, transgenic tobacco plants also displayed an enhanced tolerance to salt and drought stresses, mainly demonstrated by the transgenic lines exhibiting lower accumulation of MDA content and higher POD (EC 1.11.1.7), SOD (EC 1.15.1.1) activities, chlorophyll content, photosynthetic rate and stomatal conductance, accompanied by enhanced expression of defense-related genes (NtPOD, NtSOD, NtLEA5, NtP5CS and NtNCED1) under salt and drought stresses. Overall, these findings suggest that SpWRKY1 acts as a positive regulator involved in tobacco defense responses to biotic and abiotic stresses.

  11. Agroinoculation of a full-length cDNA clone of cotton leafroll dwarf virus (CLRDV) results in systemic infection in cotton and the model plant Nicotiana benthamiana.

    PubMed

    Delfosse, Verónica C; Casse, María F; Agrofoglio, Yamila C; Kresic, Iván Bonacic; Hopp, Horacio E; Ziegler-Graff, Véronique; Distéfano, Ana J

    2013-07-01

    Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNAs were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse

  12. Pseudomonas Type III effector AvrPto suppresses the programmed cell death induced by two nonhost pathogens in Nicotiana benthamiana and tomato.

    PubMed

    Kang, Li; Tang, Xiaoyan; Mysore, Kirankumar S

    2004-12-01

    Many gram-negative bacterial pathogens rely on a type III secretion system to deliver a number of effector proteins into the host cell. Though a number of these effectors have been shown to contribute to bacterial pathogenicity, their functions remain elusive. Here we report that AvrPto, an effector known for its ability to interact with Pto and induce Pto-mediated disease resistance, inhibited the hypersensitive response (HR) induced by nonhost pathogen interactions. Pseudomonas syringae pv. tomato T1 causes an HR-like cell death on Nicotiana benthamiana. This rapid cell death was delayed significantly in plants inoculated with P. syringae pv. tomato expressing avrPto. In addition, P. syringae pv. tabaci expressing avrPto suppressed nonhost HR on tomato prf3 and ptoS lines. Transient expression of avrPto in both N. benthamiana and tomato prf3 plants also was able to suppress nonhost HR. Interestingly, AvrPto failed to suppress cell death caused by other elicitors and nonhost pathogens. AvrPto also failed to suppress cell death caused by certain gene-for-gene disease resistance interactions. Experiments with avrPto mutants revealed several residues important for the suppression effects. AvrPto mutants G2A, G99V, P146L, and a 12-amino-acid C-terminal deletion mutant partially lost the suppression ability, whereas S94P and 196T enhanced suppression of cell death in N. benthamiana. These results, together with other discoveries, demonstrated that suppression of host-programmed cell death may serve as one of the strategies bacterial pathoens use for successful invasion. PMID:15597738

  13. A MADS-box gene NtSVP regulates pedicel elongation by directly suppressing a KNAT1-like KNOX gene NtBPL in tobacco (Nicotiana tabacum L.).

    PubMed

    Wang, Di; Chen, Xiaobo; Zhang, Zenglin; Liu, Danmei; Song, Gaoyuan; Kong, Xingchen; Geng, Shuaifeng; Yang, Jiayue; Wang, Bingnan; Wu, Liang; Li, Aili; Mao, Long

    2015-10-01

    Optimal inflorescence architecture is important for plant reproductive success by affecting the ultimate number of flowers that set fruits and for plant competitiveness when interacting with biotic or abiotic conditions. The pedicel is one of the key contributors to inflorescence architecture diversity. To date, knowledge about the molecular mechanisms of pedicel development is derived from Arabidopsis. Not much is known regarding other plants. Here, an SVP family MADS-box gene, NtSVP, in tobacco (Nicotiana tabacum) that is required for pedicel elongation was identified. It is shown that knockdown of NtSVP by RNA interference (RNAi) caused elongated pedicels, while overexpression resulted in compact inflorescences with much shortened pedicels. Moreover, an Arabidopsis BREVIPEDECELLUS/KNAT1 homologue NtBP-Like (NtBPL) was significantly up-regulated in NtSVP-RNAi plants. Disruption of NtBPL decreased pedicel lengths and shortened cortex cells. Consistent with the presence of a CArG-box at the NtBPL promoter, the direct binding of NtSVP to the NtBPL promoter was demonstrated by yeast one-hybrid assay, electrophoretic mobility shift assay, and dual-luciferase assay, in which NtSVP may act as a repressor of NtBPL. Microarray analysis showed that down-regulation of NtBPL resulted in differential expression of genes associated with a number of hormone biogenesis and signalling genes such as those for auxin and gibberellin. These findings together suggest the function of a MADS-box transcription factor in plant pedicel development, probably via negative regulation of a BP-like class I KNOX gene. The present work thus postulates the conservation and divergence of the molecular regulatory pathways underlying the development of plant inflorescence architecture. PMID:26175352

  14. PeBL1, a Novel Protein Elicitor from Brevibacillus laterosporus Strain A60, Activates Defense Responses and Systemic Resistance in Nicotiana benthamiana

    PubMed Central

    Wang, Haoqian; Yang, Xiufen; Guo, Lihua; Zeng, Hongmei

    2015-01-01

    We report the identification, characterization, and gene cloning of a novel protein elicitor (PeBL1) secreted from Brevibacillus laterosporus strain A60. Through a purification process consisting of ion-exchange chromatography and high-performance liquid chromatography (HPLC), we isolated a protein that was identified by electrospray ionization quadrupole time of flight tandem mass spectrometry (ESI–Q-TOF–MS-MS). The 351-bp PeBL1 gene produces a 12,833-Da protein with 116 amino acids that contains a 30-residue signal peptide. The PeBL1 protein was expressed in Escherichia coli. The recombinant protein can induce a typical hypersensitive response (HR) and systemic resistance in Nicotiana benthamiana, like the endogenous protein. PeBL1-treated N. benthamiana exhibited strong resistance to the infection of tobacco mosaic virus-green fluorescent protein (TMV-GFP) and Pseudomonas syringae pv. tabaci compared to control N. benthamiana. In addition, PeBL1 triggered a cascade of events that resulted in defense responses in plants, including reactive oxygen species (ROS) production, extracellular-medium alkalization, phenolic-compound deposition, and expression of several defense-related genes. Real-time quantitative-PCR analysis indicated that the known defense-related genes PR-1, PR-5, PDF1.2, NPR1, and PAL were upregulated to varying degrees by PeBL1. This research not only provides insights into the mechanism by which beneficial bacteria activate plant systemic resistance, but also sheds new light on a novel strategy for biocontrol using strain A60. PMID:25662975

  15. Proline Metabolism in the Wild-Type and in a Salt-Tolerant Mutant of Nicotiana plumbaginifolia Studied by 13C-Nuclear Magnetic Resonance Imaging1

    PubMed Central

    Roosens, Nancy H.; Willem, Rudolph; Li, Yan; Verbruggen, Ingrid; Biesemans, Monique; Jacobs, Michel

    1999-01-01

    To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. 13C-Nuclear magnetic resonance with [5-13C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mm NaCl stress in the presence of [5-13C]Glu, a significant enrichment in [5-13C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-13C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-13C]Glu into [5-13C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Δ-pyrroline-5-carboxylate synthetase enzyme. PMID:10594115

  16. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora.

    PubMed

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  17. The N-terminal fragment of the tomato torrado virus RNA1-encoded polyprotein induces a hypersensitive response (HR)-like reaction in Nicotiana benthamiana.

    PubMed

    Wieczorek, Przemysław; Obrępalska-Stęplowska, Aleksandra

    2016-07-01

    The hypersensitive response (HR) is a defence reaction observed during incompatible plant-pathogen interactions in plants infected with a wide range of fungi, bacteria and viruses. Here, we show that an N-terminal polyprotein fragment encoded by tomato torrado virus RNA1, located between the first ATG codon and the protease cofactor (ProCo) motif, induces an HR-like reaction in Nicotiana benthamiana. Agrobacterium tumefaciens-mediated transient expression of the first 105 amino acids (the calculated molecular weight of the fragment was ca. 11.33 kDa, hereafter refered to as the 11K domain) from ToTV RNA1 induced an HR-like phenotype in infiltrated leaves. To investigate whether the 11K domain could influence the virulence and pathogenicity of a recombinant virus, we created a potato virus X (PVX) with the 11K coding sequence inserted under a duplicated coat protein promoter. We found that 11K substantially increased the virulence of the recombinant virus. Disease phenotype induced in N. benthamiana by PVX-11K was characterized by strong local and systemic necrosis. This was not observed when the 11K domain was expressed from PVX in an antisense orientation. Further analyses revealed that the 11K domain could not suppress posttranscriptional gene silencing (PTGS) of green fluorescent protein (GFP) in the N. benthamiana 16c line. In silico analysis of the predicted secondary structure of the 11K domain indicated the presence of two putative helices that are highly conserved in tomato-infecting representatives of the genus Torradovirus. PMID:27072852

  18. Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines.

    PubMed

    Nelson, Guillermo; Marconi, Patricia; Periolo, Osvaldo; La Torre, José; Alvarez, María Alejandra

    2012-06-22

    The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest inductor of the immune response. There are several available commercial vaccines against bovine viral diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts. An estimated amount of 20 μg of tE2 per gram of fresh leaves was regularly obtained with this plant system. Injection of guinea pigs with plant extracts containing 20 μg of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines. This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the advantage to be free of any eventual animal contaminant.

  19. The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

    SciTech Connect

    Lim, Hyoun-Sub; Nam, Jiryun; Seo, Eun-Young; Nam, Moon; Vaira, Anna Maria; Bae, Hanhong; Jang, Chan-Yong; Lee, Cheol Ho; Kim, Hong Gi; Roh, Mark; Hammond, John

    2014-03-15

    Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP{sub SP}) with that from AltMV-Po (CP{sub Po}) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP{sub Po} [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP{sub SP} but not CP{sub Po} interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP{sub SP} than CP{sub Po} in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. - Highlights: • Alternanthera mosaic virus CP is an elicitor of systemic necrosis in N. benthamiana. • Virus-induced systemic necrosis is enhanced at 15 °C compared to 25 °C. • Induction of systemic necrosis is dependent on as few as two CP amino acid residues. • These residues are at subunit interfaces within the same turn of the virion helix. • Inducer/non-inducer CPs interact differentially with a boron transporter protein.

  20. Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana.

    PubMed

    Khakimov, Bekzod; Kuzina, Vera; Erthmann, Pernille Ø; Fukushima, Ery Odette; Augustin, Jörg M; Olsen, Carl Erik; Scholtalbers, Jelle; Volpin, Hanne; Andersen, Sven Bode; Hauser, Thure P; Muranaka, Toshiya; Bak, Søren

    2015-11-01

    The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs).

  1. Relationship between leaf antioxidants and ozone injury in Nicotiana tabacum 'Bel-W3' under environmental conditions in São Paulo, SE - Brazil

    NASA Astrophysics Data System (ADS)

    Esposito, Marisia P.; Ferreira, Mauricio L.; Sant'Anna, Silvia M. R.; Domingos, Marisa; Souza, Silvia R.

    Previous studies have reported that the extent of leaf injury in Nicotiana tabacum "Bel-W3" exposed to environmental conditions in the city of São Paulo is influenced by weather conditions. This influence may occur by means of antioxidant responses. Thus, this study aimed to evaluate whether daily antioxidant responses to environmental variations interfere on the progression of leaf injury on plants of this cultivar during their exposure in a state park of São Paulo and to determine a linear combination of variables, among antioxidants and environmental factors, which mostly explain this visible response. Plants were exposed at the mentioned site for 14 days in four different experiments. During each experiment, three plants were daily sampled to determine the accumulated percentage of leaf area affected by necrosis and antioxidant responses (concentrations of total ascorbic acid (AA) and activity of superoxide dismutase (SOD) and peroxidases (POD)). Ozone concentrations and weather conditions were also daily measured. Pearson correlations and multivariate analyses assessed the relationship between biological and environmental variables. Leaf injury appeared between the 3rd and 6th days of exposure and increased over the exposure periods. The daily concentrations of AA tended to decrease with time of exposure in all experiments, but the activity of SOD and POD oscillated during plant exposure. Positive correlations were observed between AA or SOD and O 3 concentrations, as well as negative correlations between AA and air temperature. The increasing percentage of leaf necrosis across the whole period was explained by decreasing levels of AA 2 days before injury estimation and by higher O 3 concentrations 5 days before ( R2 = 0.36; p < 0.001). The use of N. tabacum Bel-W3 as a bioindicator can be restricted by leaf antioxidant responses to both atmospheric contamination and weather conditions.

  2. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

    PubMed Central

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  3. DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana contributes to potyvirus movement and transports to plasmodesmata via the early secretory pathway and the actomyosin system.

    PubMed

    Geng, Chao; Cong, Qian-Qian; Li, Xiang-Dong; Mou, An-Li; Gao, Rui; Liu, Jin-Liang; Tian, Yan-Ping

    2015-02-01

    The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 5'-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement.

  4. Deep Sequencing–Based Transcriptome Profiling Reveals Comprehensive Insights into the Responses of Nicotiana benthamiana to Beet necrotic yellow vein virus Infections Containing or Lacking RNA4

    PubMed Central

    Fan, Huiyan; Sun, Haiwen; Wang, Ying; Zhang, Yongliang; Wang, Xianbing; Li, Dawei; Yu, Jialin; Han, Chenggui

    2014-01-01

    Background Beet necrotic yellow vein virus (BNYVV), encodes either four or five plus-sense single stranded RNAs and is the causal agent of sugar beet rhizomania disease, which is widely distributed in most regions of the world. BNYVV can also infect Nicotiana benthamiana systemically, and causes severe curling and stunting symptoms in the presence of RNA4 or mild symptoms in the absence of RNA4. Results Confocal laser scanning microscopy (CLSM) analyses showed that the RNA4-encoded p31 protein fused to the red fluorescent protein (RFP) accumulated mainly in the nuclei of N. benthamiana epidermal cells. This suggested that severe RNA4-induced symptoms might result from p31-dependent modifications of the transcriptome. Therefore, we used next-generation sequencing technologies to analyze the transcriptome profile of N. benthamiana in response to infection with different isolates of BNYVV. Comparisons of the transcriptomes of mock, BN3 (RNAs 1+2+3), and BN34 (RNAs 1+2+3+4) infected plants identified 3,016 differentially expressed transcripts, which provided a list of candidate genes that potentially are elicited in response to virus infection. Our data indicate that modifications in the expression of genes involved in RNA silencing, ubiquitin-proteasome pathway, cellulose synthesis, and metabolism of the plant hormone gibberellin may contribute to the severe symptoms induced by RNA4 from BNYVV. Conclusions These results expand our understanding of the genetic architecture of N. benthamiana as well as provide valuable clues to identify genes potentially involved in resistance to BNYVV infection. Our global survey of gene expression changes in infected plants reveals new insights into the complicated molecular mechanisms underlying symptom development, and aids research into new strategies to protect crops against viruses. PMID:24416380

  5. A proteinase inhibitor from Nicotiana alata inhibits the normal development of light-brown apple moth, Epiphyas postvittana in transgenic apple plants.

    PubMed

    Maheswaran, Gowri; Pridmore, Lucinda; Franz, Peter; Anderson, Marilyn A

    2007-06-01

    Insecticidal proteins are a potential resource to enhance resistance to insect pests in transgenic plants. Here, we describe the generation and analysis of the apple cultivar 'Royal Gala' transgenic for Nicotiana alata (N. alata) proteinase inhibitor (PI) and the impact of this PI on the growth and development of the Epiphyas postvittiana (light-brown apple moth). A cDNA clone encoding a proteinase inhibitor precursor from N. alata (Na-PI) under the control of either a double 35S promoter or a promoter from a ribulose-1,5-bisphosphate carboxylase small sub-unit gene (rbcS-E9 promoter) was stably incorporated into 'Royal Gala' apple using Agrobacterium-mediated transformation. A 40.3 kDa Na-PI precursor protein was expressed and correctly processed into 6-kDa proteinase inhibitors in the leaves of transgenic apple lines. The 6-kDa polypeptides accumulated to levels of 0.05 and 0.1% of the total soluble protein under the control of the rbc-E9 promoter and the double 35S promoter, respectively. Light-brown apple moth larvae